Autism-associated biomarkers and uses thereof

ABSTRACT

The invention discloses biomarkers for human autism. The invention provides methods for treating, preventing, and diagnosing human autism and autism-related disorders.

This application is a continuation-in-part of International ApplicationNumber PCT/US2010/034254, filed on May 10, 2010, the contents of whichare hereby incorporated by reference in its entirety. This applicationalso claims priority to U.S. Provisional Application No. 61/527,313filed on Aug. 25, 2011, the contents of which are hereby incorporated byreference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. U01NS047537 and Grant No. AI57158 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 14, 2012, isnamed 1924849U.txt and is 112,136 bytes in size.

BACKGROUND OF THE INVENTION

Autistic disorder is one of five pervasive developmental disordersdefined in the Diagnostic and Statistical Manual of Mental Disorders,Fourth Edition, Text Revision DSM-IV-TR (2000). Autistic disorder is adevelopmental disorder of the human brain that manifests during infancyor childhood and is characterized by behavioral and social abnormalitiesthat appear to be developmentally based (for example, impairments insocial interaction and communication). In addition, autism interfereswith imagination and the ability to reason. Autism is frequentlyassociated with other disorders such as attention deficit/hyperactivitydisorder (AD/HD) and can be associated with psychiatric symptoms such asanxiety and depression. In the last decade, autism diagnoses haveincreased by 300% to 500% in the United States and many other countries.A means of prevention and treatment is needed for this health crisisthat addresses the underlying mechanisms leading to the development ofautism versus those that merely address the symptoms.

Pervasive developmental disorders (PDDs) are also part of the AutismSpectrum Disorders (ASDs). PDD is used to categorize children who do notmeet the strict criteria for Autistic Disorder but who come close,either by manifesting atypical autism or by nearly meeting thediagnostic criteria in two or three of the key areas. Some of thesechildren meet criteria for the ASD known as Asperger's Disorder (ASP),wherein language capacities are relatively spared compared to childrenwith Autistic Disorder. Others meet criteria for the PDDs known asChildhood Disintegrative Disorder, which begins at a slightly later agethan the other ASDs, or Rett's Disorder, which is related to a mutationin a DNA methylation binding protein gene called MeCP2 and usuallyoccurs in girls.

Many children with autism have gastrointestinal (GI) disturbances thataffect their quality of life. Although some of these children have beeninvestigated through GI immunopathology, molecular studies are lackingthat characterize host gene expression or survey microflora usingpyrosequencing methods.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the finding that decreasedlevels in sucrase isomaltase, maltase glucoamylase, lactase, GLUT2, andSGLT1 can serve as markers for human Autism Spectrum Disorders.Accordingly, in one aspect, the invention provides a method fordetecting the presence of or a predisposition to autism or an autismspectrum disorder (ASD) in a human subject or a child of a humansubject. The method comprises: (1) obtaining a biological sample from ahuman subject; and (2) detecting whether or not there is an alterationin the expression of a carbohydrate metabolic enzyme protein or acarbohydrate transporter protein in the subject as compared to anon-autistic subject. In one embodiment, the carbohydrate metabolicenzyme comprises sucrase isomaltase, maltase glucoamylase, lactase, or acombination thereof. In another embodiment, the carbohydrate transportercomprises GLUT2, SGLT1, or a combination thereof. In some embodiments,the method further comprises detecting a decrease in Bacteriodetes, anincrease in the Firmicute/Bacteroidete ratios, an increase in cumulativelevels of Firm icutes and Proteobacteria, an increase inBeta-proteobacteria, and an increase in Sutterella sp. in the small orlarge intestine of the subject. In one embodiment, the detectingcomprises detecting whether there is an alteration in the gene locusthat encodes the carbohydrate metabolic enzyme protein or thecarbohydrate transporter protein. In a further embodiment, the detectingcomprises detecting whether expression of the carbohydrate metabolicenzyme protein or the carbohydrate transporter protein is reduced. Insome embodiments, the detecting comprises detecting in the samplewhether there is a reduction in the mRNA expression of the carbohydratemetabolic enzyme protein or the carbohydrate transporter protein. Insome embodiments of the invention, the subject is a human embryo, ahuman fetus, or an unborn human child. In other embodiments, the samplecomprises blood, serum, sputum, lacrimal secretions, semen, vaginalsecretions, fetal tissue, skin tissue, small intestine tissue (e.g., theileum), large intestine tissue (e.g., the cecum), muscle tissue,amniotic fluid, or a combination thereof.

An aspect of the invention provides a method for treating or preventingautism or an autism spectrum disorder in a subject in need thereof. Themethod comprises administering to the subject a therapeutic amount of apharmaceutical composition comprising a functional carbohydratemetabolic enzyme molecule or a carbohydrate transporter molecule,thereby treating or preventing autism or an autism spectrum disorder. Ina further embodiment, the administering comprises a subcutaneous,intra-muscular, intra-peritoneal, or intravenous injection; an infusion;oral, nasal, or topical delivery; or a combination of the delivery modesdescribed. In some embodiments, the administering comprises delivery ofa carbohydrate metabolic enzyme molecule or a carbohydrate transportermolecule to the alimentary canal or intestine of the subject. In otherembodiments, the administering comprises feeding the human subject orchild thereof a therapeutically effective amount of the carbohydratemetabolic enzyme molecule or a carbohydrate transporter molecule. Infurther embodiments, the administering occurs daily, weekly, twiceweekly, monthly, twice monthly, or yearly.

In other aspects, the invention provides for a pharmaceuticalcomposition comprising: a carbohydrate metabolic enzyme molecule or acarbohydrate transporter molecule molecule; and a pharmaceuticallyacceptable carrier.

An aspect of the invention provides for an isolated nucleic acidcomposition. In one embodiment, the composition comprises a nucleic acidmolecule having at least about 80% identity to SEQ ID NO: 11, 12, 13, or14. In one embodiment, the composition comprises a nucleic acid moleculehaving at least about 85% identity to SEQ ID NO: 11, 12, 13, or 14. Inone embodiment, the composition comprises a nucleic acid molecule havingat least about 90% identity to SEQ ID NO: 11, 12, 13, or 14. In oneembodiment, the composition comprises a nucleic acid molecule having atleast about 95% identity to SEQ ID NO: 11, 12, 13, or 14. In oneembodiment, the composition comprises a nucleic acid molecule having atleast about 98% identity to SEQ ID NO: 11, 12, 13, or 14. In oneembodiment, the composition comprises a nucleic acid molecule having atleast about 99% identity to SEQ ID NO: 11, 12, 13, or 14. In oneembodiment, the composition is SEQ ID NO: 11, 12, 13, or 14.

An aspect of the invention provides for a diagnostic kit for detectingthe presence of Sutterella sp. in a sample. In one embodiment, the kitcomprises a nucleic acid molecule that specifically hybridizes to or aprimer combination that amplifies a Sutterella sp. 16S nucleic acidsequence. In one embodiment, the nucleic acid molecule comprises anucleic acid primer or nucleic acid probe. In another embodiment, the16S nucleic acid sequence comprises at least about 80% of SEQ ID NO: 59or SEQ ID NO: 60. In some embodiments, the 16S nucleic acid sequencecomprises at least about 85% of SEQ ID NO: 59 or SEQ ID NO: 60. Infurther embodiments, the 16S nucleic acid sequence comprises at leastabout 90% of SEQ ID NO: 59 or SEQ ID NO: 60. In other embodiments, the16S nucleic acid sequence comprises at least about 95% of SEQ ID NO: 59or SEQ ID NO: 60. In another embodiment, the 16S nucleic acid sequencecomprises at least about 98% of SEQ ID NO: 59 or SEQ ID NO: 60. In someembodiments, the 16S nucleic acid sequence comprises at least about 99%of SEQ ID NO: 59 or SEQ ID NO: 60. In further embodiments, the 16Snucleic acid sequence is SEQ ID NO: 59 or SEQ ID NO: 60. In oneembodiment, the probe comprises a nucleotide sequence having SEQ ID NOS:13 or 14 in Table 1, or the italicized nucleotide of sequence SEQ ID NO:19. In a further embodiment, the probe comprises at least 10 consecutivenucleotide bases comprising SEQ ID NO: 19, wherein S is a G nucleotideand/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide,wherein R is an A nucleotide and/or G nucleotide, wherein W is an Anucleotide and/or T nucleotide, and wherein H is an A nucleotide and/orT nucleotide and/or C nucleotide. In some embodiments, the probecomprises a reverse complement of SEQ ID NOS: 11, 12, 15, 16, 17, 18, or19, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a Cnucleotide and/or T nucleotide, wherein R is an A nucleotide and/or Gnucleotide, wherein W is an A nucleotide and/or T nucleotide, andwherein H is an A nucleotide and/or T nucleotide and/or C nucleotide. Inother embodiments, the primer comprises a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 11, 12, 15, 16, 17, or 18,wherein, wherein S is a G nucleotide and/or a C nucleotide, wherein Y isa C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/orG nucleotide, wherein W is an A nucleotide and/or T nucleotide, andwherein H is an A nucleotide and/or T nucleotide and/or C nucleotide. Inone embodiment, the sample is from a human or non-human animal. In otherembodiments, the sample comprises intestinal tissue (e.g., the smallintestine or large intestine), feces, blood, skin, or a combination ofthe mentioned tissues.

An aspect of the invention provides for a diagnostic kit for determiningwhether a sample from a subject exhibits a presence of or apredisposition to autism or an autism spectrum disorder (ASD). In oneembodiment, the kit comprising a nucleic acid primer that specificallyhybridizes to an autism biomarker, wherein the primer will prime apolymerase reaction only when an autism biomarker is present. In anotherembodiment, the primer comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 11, 12, 15, 16, 17, or 18, wherein,wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a Cnucleotide and/or T nucleotide, wherein R is an A nucleotide and/or Gnucleotide, wherein W is an A nucleotide and/or T nucleotide, andwherein H is an A nucleotide and/or T nucleotide and/or C nucleotide. Insome embodiments, the autism biomarker is a carbohydrate trasportermolecule, a carbohydrate metabolic enzyme molecule, or agastrointestinal Sutterella sp. bacterium. In a further embodiment, thecarbohydrate trasporter molecule is GLUT2 or SGLT1. In otherembodiments, the carbohydrate metabolic enzyme molecule is SI, MGAM, orLCT. In one embodiment, the sample is from a human or non-human animal.In other embodiments, the sample comprises intestinal tissue (e.g., thesmall intestine or large intestine), feces, blood, skin, or acombination of the mentioned tissues.

An aspect of the invention provides for a method of treating orpreventing a disease associated with elevated levels ofBeta-proteobacteria. The method of the invention comprises administeringto a subject in need thereof a therapeutic amount of an antimicrobialcomposition effective against Beta-proteobacteria for treating thedisease. In one embodiment, the antimicrobial composition is anantibiotic, a probiotic agent, or a combination thereof. In anotherembodiment, the disease is ASD, autism, or a gastrointestinal disease.In a further embodiment, the gastrointestinal disease is diarrhea,inflammatory bowel disease, antimicrobial-associated colitis, orirritable bowel syndrome. In some embodiments, the diarrhea orinflammatory bowel diseases is ulcerative colitis or Crohn's disease. Inone embodiment, the antibiotic comprises lincosamides, chloramphenicols,tetracyclines, aminoglycosides, beta-lactams, vancomycins, bacitracins,macrolides, amphotericins, sulfonamides, methenamin, nitrofurantoin,phenazopyridine, trimethoprim; rifampicins, metronidazoles, cefazol ins,lincomycin, spectinomycin, mupirocins, quinolones, novobiocins,polymixins, gramicidins, antipseudomonals, or a combination of thestated antibiotics. In another embodiment of the invention, theprobiotic agent comprises Bacteroides, Prevotella, Porphyromonas,Fusobacteriuni, Sutterella, Bilophila, Campylobacter, Wolinella,Butyrovibrio, Megamonas, Desulfomonas, Desulfovibrio, Bifidobacterium,Lactobacillus, Eubacterium, Actinomyces, Eggerthel la, Coriobacterium,Propionibacterium, other genera of non-sporeforming anaerobicgram-positive bacilli, Bacillus, Peptostreptococcus, newly createdgenera originally classified as Peptostreptococcus, Peptococcus,Acidaminococcus, Ruminococcus, Megasphaera, Gaffkya, Coprococcus,Veillonella, Sarcina, Clostridium, Aerococcus, Streptococcus,Enterococcus, Pediococcus, Micrococcus, Staphylococcus, Corynebacterium,species of the genera comprising the Enterobacteriaceae andPseudomonadaceae, or a combination of the listed probiotic agents.

An aspect of the invention provides for a method of detecting aSutterella sp. in a sample. The method comprises: (a) selecting aSutterlla sp.-specific primer pair, wherein the primer pair mediatesamplification of a polynucleotide amplicon of a selected, known lengthfrom a nucleic acid of a Sutterlla sp.; contacting a nucleic acid fromthe sample with the Sutterlla sp.-specific primer pair in a reactionmixture under conditions that promote amplification of a polynucleotideamplicon, wherein the primer pair will prime a polymerase reaction onlywhen the nucleic acid of a Sutterlla sp. is present; and detecting theamplicons, wherein the detection of an amplicon of a selected, knownlength is indicative of the sample containing the nucleic acid of aSutterlla sp. In one embodiment, the sample comprises intestinal tissue(e.g., the small intestine or large intestine), feces, blood, skin, or acombination of the listed tissues. In one embodiment, the primer paircomprises a forward primer and a reverse primer. In some embodiments,the forward primer comprises SEQ ID NO: 11 or 17, wherein S is a Gnucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or Tnucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein Wis an A nucleotide and/or T nucleotide, and wherein H is an A nucleotideand/or T nucleotide and/or C nucleotide. In other embodiments, thereverse primer comprises SEQ ID NO: 12 or 18, wherein S is a Gnucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or Tnucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein Wis an A nucleotide and/or T nucleotide, and wherein H is an A nucleotideand/or T nucleotide and/or C nucleotide. In further embodiments, theforward primer comprises at least 10 consecutive nucleotide basescomprising SEQ ID NO: 17 or19, wherein S is a G nucleotide and/or a Cnucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein Ris an A nucleotide and/or G nucleotide, wherein W is an A nucleotideand/or T nucleotide, and wherein H is an A nucleotide and/or Inucleotide and/or C. In some embodiments, the reverse primer comprisesat least 10 consecutive nucleotide bases comprising SEQ ID NO: 18 or19,wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a Cnucleotide and/or T nucleotide, wherein R is an A nucleotide and/or Gnucleotide, wherein W is an A nucleotide and/or T nucleotide, andwherein H is an A nucleotide and/or T nucleotide and/or C nucleotide,wherein B is a T nucleotide, C nucleotide, or G nucleotide, wherein V isan A nucleotide, G nucleotide, or C nucleotide; wherein D is an Anucleotide, G nucleotide, or T nucleotide; and wherein K is a Gnucleotide or T nucleotide.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic depicting carbohydrate metabolizing enzymes (e.g.,sucrase isomaltase, maltase glucoamylase, and lactase) and carbohydratetransporter proteins (e.g., GLUT2 and SGLT1) involved in carbohydratemetabolism, uptake, and absorption in the enterocytes of the ileum.

FIG. 2 shows bar graphs depicting that carbohydrate metabolizing enzymemRNAs are reduced in the ileum of ASD subjects. Graphs are shown forsucrase isomaltase (left), maltase glucoamylase (center), and lactase(right).

FIG. 3 shows bar graphs depicting that carbohydrate transporter mRNAsare reduced in the ileum of ASD subjects. Graphs are shown for SGLT1(Top) and GLUT2 (Bottom).

FIG. 4 shows graphs depicting that mRNA for ileal inflammatory markersare increased in the ileum of ASD subjects. Graphs are shown for C1QA(Top Left), Resistin (Top Right), and IL17F (Bottom Left and Right).

FIG. 5 shows bar graphs depicting the differences in bacteria phylumfound in the ileum of ASD subjects. Changes at the phylum level wereobserved. Bar graphs show a decrease in Bacteroidetes (left) andincrease in Firmicute/Bacteroidete ratios in ileum of AUT-GI children.

FIG. 6 is a bar graph depicting the copy number of bacteroidetes foundin the ileum of ASD subjects. Real-time PCR confirmed a decrease inBacteroidete. Bacteroidete 16S rDNA copies (Normalized to TotalBacterial 16S rDNA).

FIG. 7 is a schematic summarizing the interplay between expressionlevels of carbohydrate metabolic enzymes (e.g., sucrase isomaltase,maltase glucoamylase, and lactase), carbohydrate transporters (e.g.,GLUT2 and SGLT1) and the population of bacteria in the ileum of ASDsubjects.

FIGS. 8A-B show the Presence of Sutterella sequences in a subset ofAUT-GI patients: Detection by pyrosequencing of the V2-region of the 16SrRNA gene. FIGS. 8A-B are bar graphs showing the abundance of Sutterellasp. in the ileum (FIG. 8A) and cecum (FIG. 8B) of autism and controlpatients. Distribution of Sutterella sequences as a percentage of totalbacterial 16S rRNA gene reads from ileal (FIG. 8A; Mann-Whitney, tiedp=0.022) and cecal (FIG. 8B; Mann-Whitney, tied p =0.037) biopsies fromAUT-GI and Control-GI patients.

FIGS. 8C-D show the presence of Sutterella sequences in a subset ofAUT-GI patients: Detection by pyrosequencing of the V2-region of the 16SrRNA gene. FIGS. 8C-D are bar graphs showing the abundance of Sutterellasp. sequences in the ileum (FIG. 8C) and cecum (FIG. 8D) of autism andcontrol patients. FIGS. 8C-D shows the distribution of Sutterellasequences by individual patient as a percentage of total bacteria 16SrRNA reads from ileal (FIG. 8C) and cecal (FIG. 8D) biopsies from AUT-GI(patients #1-15) and Control-GI (patients #16-22) patients. *, p<0.05.

FIGS. 8E-F show the presence of Sutterella sequences in a subset ofAUT-GI patients: Detection by pyrosequencing of the V2-region of the 16SrRNA gene. FIGS. 8E-F are bar graphs showing the abundance of Sutterellasp. sequences comprising the Beta-proteobacteria sequences in the ileum(FIG. 8E) and cecum (FIG. 8F) of autism and control patients. FIGS. 8E-Fshow the distribution of Sutterella sequences by individual patient as apercentage of total Betaproteobacteria 16S rRNA reads from ileal (FIG.8E) and cecal (FIG. 8F) biopsies from AUT-GI (patients #1-15) andControl-GI (patients #16-22) patients. *, p<0.05.

FIG. 9 is a photograph of an agarose gel showing the results ofclassical PCR experiments for the detection of Sutterella.Sutterella-specific 16S rRNA gene (V6-V8) PCR amplification of 10-folddilutions of Sutterella plasmid DNA standards spiked into ileal DNA froma Sutterella-negative Control-GI patient. Note linear amplification downto 5×10² copies and an endpoint detection limit of 5×10¹ copies

FIG. 10A is an amplification plot of Sutterella sp. through cycles ofReal-time PCR experiments. The figure depicts Real-time PCRamplification plot of 10-fold serial dilutions of Sutterella plasmid DNAstandards.

FIG. 10B is a standard curve graph showing the copy number of Sutterellasp. from Real-time PCR experiments.

FIG. 11 is a photograph of an agarose gel showing the results ofSutterella detection in the ileum and cecum of patients using the V6-V8Sutterella sp.-specific PCR.

FIGS. 12A-B are bar graphs showing the copy number of Sutterella sp. inthe ileum (FIG. 12A) and cecum (FIG. 12B) of autism and control patientsusing the V6-V8 Sutterella sp.-specific PCR.

FIGS. 12C-D are bar graphs showing the copy number of Sutterella sp. inthe ileum (FIG. 12C) and cecum (FIG. 12D) of autism and control patientsusing the V6-V8 Sutterella sp.-specific PCR.

FIG. 13 is a sequence alignment for the V6-V8 region of Sutterella sp.obtained from biological samples of Autism patients 1, 3, 10, 11, and 12(SEQ ID NO: 59), and Autism patients 5 and 7 (SEQ ID NO: 60).

FIG. 14 depicts Sutterella sp. sequence clustering from the OperationalTaxonomic Unit (OTU) analysis of V2 pyrosequencing reads.

FIG. 15A is a schematic depicting Sutterella sp. treeing analysis of theV6-V8 sequences.

FIG. 15B is a schematic depicting Sutterella sp. treeing analysis of theV2 sequence.

FIG. 16 shows graphs of quantitative real-time PCR analysis ofdisaccharidases, hexose transporters, villin and CDX2 transcripts.Box-and-whisker plots displaying (FIG. 16A) SI (Mann-Whitney; p=0.001),(FIG. 16B) MGAM (Mann-Whitney; p=0.003), (FIG. 16C) LCT (Mann-Whitney;p=0.032), (FIG. 16D) SGLT1 (Mann-Whitney; p=0.008), (FIG. 16E) GLUT2(Mann-Whitney; p=0.010), (FIG. 16F) Villin (Mann-Whitney; p=0.307), and(FIG. 16G) CDX2 (Mann-Whitney; p=0.192) mRNA expression normalized toGAPDH mRNA in ileal biopsies from AUT-GI (AUT) and Control-GI (Control)patients. Box-and-whisker plots show the median and the interquartile(midspread) range (boxes containing 50% of all values), the whiskers(representing the 25^(th) and 75^(th) percentiles) and the extreme datapoints (open circles). *p<0.05; **, p<0.01; n.s., not significant.

FIG. 17 shows graphs depicting pyrosequencing analysis of intestinalmicrobiota in AUT-GI children. (FIGS. 17A-B) Phylum-level comparison ofthe average relative abundance of bacterial taxa in ileal (FIG. 17A) andcecal (FIG. 17B) biopsies from AUT-GI and Control-GI patients. (FIGS.17C-D) Box-and-whisker plot displaying the distribution of Bacteroidetesas a percentage of total bacterial 16S rRNA V2 pyroseqeuncing reads fromileal (C; Mann-Whitney, p=0.012) and cecal (FIG. 17D; Mann-Whitney,p=0.008) biopsies from AUT-GI and Control-GI patients. (FIGS. 17E-F)Bacteroidete-specific quantitative real-time PCR analysis of ileal (FIG.17E; Mann-Whitney, p=0.003) and cecal (FIG. 17F; Mann-Whitney, p=0.022)biopsies from AUT-GI and Control-GI patients. (FIGS. 17G-H) Heatmapsdisplaying abundance distributions (% of total sequence reads perpatient) of Bacteroidetes classified at the family level in ileal (FIG.17G) and cecal (FIG. 17H) biopsies from AUT-GI and Control-GI children(Bottom row displays cumulative levels of all family members bypatient). copy number values are normalized relative to total bacteriacopy numbers; *,p<0.05, **,p<0.01.

FIG. 18 shows graphs of Firmicute abundance in AUT-GI and Control-GIchildren. (FIGS. 18A-18B) Box-and-whisker plots displaying theFirmicute/Bacteroidete ratio from pyrosequencing reads obtained fromileal (FIG. 18A; Mann-Whitney, p=0.026) and cecal (FIG. 18B;Mann-Whitney, p=0.032) biopsies of AUT-GI and Control-GI patients.(FIGS. 18C-18D) Box-and-whisker plots displaying the cumulative levelsof members of the families Lachnospiraceae and Ruminococcaceae in ileal(FIG. 18C; Mann-Whitney; p=0.062) and cecal (FIG. 18D; Mann-Whitney;p=0.098) biopsies from AUT-GI and Control-GI children. (FIGS. 18E-18F)Heatmaps displaying abundance distribution (% of total sequence readsper patient) of family members in the class Clostridia in ileum (FIG.18E) and cecum (FIG. 18F) of AUT-GI and Control-Gi children (Bottom rowdisplays cumulative levels of all family members by patient). (FIGS.18G-18H) Box-and-whisker plots displaying the cumulative abundance ofFirmicutes and Proteobacteria from ileal (FIG. 18G; Mann-Whitney,p=0.015) and cecal (FIG. 18H; Mann-Whitney, p=0.007) biopsies fromAUT-GI and Control-GI patients. (FIGS. 18I-18J) Heatmaps displaying theabundance distribution (% of total sequence reads per patient) ofFirmicutes and Proteobacteria by patient in ilea (FIG. 18I) and ceca(FIG. 18J) of AUT-GI and Control-GI children (Bottom row displayscumulative levels of Firmicutes and Proteobacteria by patient).*,p<0.05, **,p<0.01, †, p<0.1 (trend).

FIG. 19 shows graphs of the abundance of Proteobacteria in AUT-GI andControl-GI children. (FIGS. 19A-19B) Box-and-whisker plots displayingthe phyla level abundance of Proteobacteria members in ilea (FIG. 19A;Mann-Whitney, p=0.549) and ceca (FIG. 19B; Mann-Whitney, p=0.072) ofAUT-GI and Control-GI children biopsies obtained by pyrosequencing.(FIGS. 19C-19D) Box-and-whisker plots displaying the class levelabundance of Betaproteobacteria members in ilea (FIG. 19C; Mann-Whitney,p=0.072) and ceca (FIG. 19D; p=0.038) of AUT-GI and Control-GI children.(FIGS. 19E-19F) Heatmaps displaying the abundance distribution (% oftotal sequence reads per patient) of family members within the classesAlpha-, Beta-, and Gammaproteobacteria in the ilea (FIG. 19E) and ceca(FIG. 19F) of AUT-GI and Control-GI children (Bottom row of each heatmapdisplays the cumulative levels of family members in each class bypatient). *,p<0.05, †, p<0.1 (trend); n.s., not significant.

FIG. 20 shows schematics depicting factors that mediate GI disease inAUT-GI children. (FIG. 20A) Schematic representation ofenterocyte-mediated digestion of disaccharides and absorption/transportof monosaccharides in the small intestine. Disaccharidase enzymes (SI,MGAM, and LCT) in the enterocyte brush border break down disaccharidesinto their component monosaccharides. The monosaccharides, glucose andgalactose, are transported from the small intestinal lumen into theenterocyte by the sodium-dependent transporter SGLT1. On the basolateralenterocyte membrane, the facilitative transporter, GLUT2, transportsglucose, galactose and fructose out of the enterocyte and into thecirculation, thus regulating postprandial blood glucose levels. GLUT2can also be transiently inserted into the apical enterocyte membrane,contributing a diffusive component to monosaccharide absorption incertain circumstances (Kellet et al., 2008). The expression levels ofdisaccharidases and hexose transporters can be controlled by thetranscription factor CDX2. (FIG. 20B) In the normal small intestine,where expression of disaccharidases and hexose transporters are high,the majority, if not all, of disaccharides are efficiently digested andmonosaccharides are absorbed from the lumen. Thus, only complexpolysaccharides reach the large intestine and serve as growth substratesfor colonic bacteria. Those bacteria best suited for growth onpolysaccharides (i.e., Bacteroidetes) outcompete other bacteria anddominate the colonic space. In the normal intestine, colonic (i.e.,cecal) microbial community structure can be kept within a normalhomeostatic range by the level of expression of disaccharidases andhexose transporters upstream in the small intestine. The constraint onbacterial structure regulated by ileal gene expression would constrainbacterial byproducts of fermentation such as SCFAs, and limit the growthof potential pathogens. (FIG. 20C) In the AUT-GI intestine, whereexpression of disaccharidases and hexose transporters are deficient,mono- and disaccharides accumulate in the lumen of the distal smallintestine (ileum) and proximal colon (cecum), and can exertextraintestinal effects by reducing postprandial blood glucose. Thepresence of additional carbohydrate substrates in the lumen abrogatesthe growth advantage of bacteria best suited for growth onpolysaccharides (i.e., Bacteroidetes) and promotes the growth of otherbacteria. In ASD-GI this specifically manifests as an increase inFirmicute/Bacteroidete ratios, cumulative levels of Firmicutes andProteobacteria, and in levels of Betaproteobacteria in both the ileumand cecum. The level of dysbiosis in the ileum and cecum can thus becontrolled by the degree and type of deficiency of carbohydratemetabolism and transport in the small intestine. Within the intestine,malabsorbed monosaccharides can lead to osmotic diarrhea; non-absorbedsugars can also serve as substrates for intestinal microflora, thatproduce fatty acids and gases (methane, hydrogen, and carbon dioxide)and promote additional GI symptoms of bloating and flatulence.Additional effects of dysbiosis can manifest in changes in SCFAs thatcan reduce colonic pH, further inhibiting the growth of Bacteroidetes.Disruption of symbiotic relationships between the host and theintestinal microbial ecosystem as a result of dysbiosis can also play afundamental role in development, distribution, activation anddifferentiation of immune cells within the intestine (Abt and Artis,2009; Mazmanian et al., 2008), thus providing a framework forunderstanding previous reports of inflammatory indices in the AUT-GIintestine.

FIG. 21 depicts lactase genotyping. (FIG. 21A) Representative agarosegel banding patterns observed for LCT-13910 and LCT-22018 polymorphisms.(FIG. 21B) Distribution of genotypes for 13910 and 22018 polymorphismsbetween AUT-GI (n=15) and Control-GI (n=7) patients (chi-squared test,p=0.896). (FIG. 21C) Box-and-whisker plot displaying the distribution ofLCT mRNA expression in all individuals (AUT-GI and Control-GI) with thehomozygous adult-type hypolactasia genotype (13910-C/C; 22018G/G)compared to all individuals (AUT-GI and Control-GI) possessing at leastone copy of the normal allele (heterozygous: 13910-C/T; 22018-G/A andhomozygous: 13910-T/T; 22018-A/A); Mann-Whitney, p=0.033. (FIG. 21D)Distribution of LCT mRNA expression levels split by genotype and group(AUT-GI and Control-GI); Kruskal-Wallis,p=0.097. (FIG. 21E) Distributionof LCT mRNA expression for all patients possessing at least one copy ofthe normal (lactase persistence) allele for AUT-GI (n=12) and Control-GI(n=6); Mann-Whitney, p=0.0246. Adult-type hypolactasia genotype ishighlighted in red. *, p<0.05.

FIG. 22 shows graphs depicting Villin normalization and CDX2 expressionstratified by total disaccharidase and transporter deficiencies.Disaccharidase or transporter mRNA/villin mRNA ratio for SI (FIG. 22A;Mann-Whitney, p=0.001), MGAM (FIG. 22B; Mann-Whitney, p=0.001), LCT(FIG. 22C; Mann-Whitney, p=0.005), SGLT1 (FIG. 22D; Mann-Whitney,p=0.0008), and GLUT2 (FIG. 22E; Mann-Whitney, p=0.002). *,p<0.05,**,p<0.01, ***,p<0.001;†, p<0.1 (trend).

FIG. 23 shows graphs of the diversity of AUT-GI and Control-GIphylotypes. (FIGS. 23A-23B) Rarefaction curves assessing thecompleteness of sampling from pyrosequencing data obtained forindividual AUT-GI (red) and Control-GI (blue) subjects' ileal (FIG. 23A)and cecal (FIG. 23B) biopsies. The y-axis indicates the number of OTUsdetected (defined at 97% threshold for sequence similarity), the x-axisindicates the number of sequences sampled. (FIGS. 23C-23D) Rarefactioncurves to estimate phylotype diversity, using the Shannon DiversityIndex, from pyrosequencing data obtained for individual AUT-GI (red) andControl-GI (blue) subjects' ileal (FIG. 23C) and cecal (FIG. 23D)biopsies.

FIG. 24 shows graphs depicting the distribution of pyrosequencing readsby patient. (FIGS. 24A-24B) Phylum level distribution of bacteria bypatient obtained from 16S rRNA gene barcoded pyrosequencing for ilea(FIG. 24A) and ceca (FIG. 24B). (FIGS. 24C-D) Distribution of lowabundance bacterial phyla obtained by barcoded pyroseqeuncing.By-patient distribution of low abundance bacterial phyla in ilea (FIG.24C) and ceca (FIG. 24D) from AUT-GI (patients 1-15) and Control-GI(patients 16-22).

FIG. 25 shows the OTU analysis of Bacteroidete phylotypes. (FIGS.25A-25B) Heatmaps displaying abundance distributions (% of totalsequence reads per patient) of the 12 most abundant Bacteroidete OTUs(defined at 97% threshold) in ileal (FIG. 25A) and cecal (FIG. 25B)biopsies from AUT-GI and Control-GI children (Bottom row displayscumulative levels of all 12 OTUs by patient). (FIGS. 25C-25D)Box-and-whisker plots displaying the cumulative abundance of the 12 OTUsin ilea (FIG. 25C; Mann-Whitney, p=0.008) and ceca (FIG. 25D;Mann-Whitney, p=0.008) of AUT-GI and Control-GI children. (FIG. 25E)Greengenes- or microbial blast(*)-derived classification ofrepresentative sequences obtained from each Bacteroidete OTU. Color codedenotes the family-level, Ribosomal Database-derived taxonomicclassification of each representative OTU sequence. **, p<0.01

FIG. 26 shows graphs depicting order-level analysis. ofFirmicute/Bacteroidete ratio and confirmation by real-time PCR. (FIGS.26A-26B) Box-and-whisker plot displaying the order-level distribution ofthe Clostridiales/Bacteroidales ratio from pyrosequencing reads obtainedfrom ileal (FIG. 26A; Mann-Whitney, p=0.012) and cecal (FIG. 26B;Mann-Whitney, p=0.032) biopsies from AUT-GI and Control-GI patients.(FIGS. 26C-26D) Box-and-whisker plot displaying theFirmicute/Bacteroidete ratios obtained by real-time PCR for ilea (FIG.26C; Mann-Whitney, p=0.0006) and ceca (FIG. 26D; Mann-Whitney, p=0.022)of AUT-GI and Control-GI children. *, p<0.05, ***, p<0.001.

FIG. 27 shows graphs of the abundance of Firmicutes assayed bypyrosequencing and real-time PCR. (FIGS. 27A-27B) Box-and-whisker plotsdisplaying the phyla level abundance of Firmicutes in the ilea (FIG.27A; Mann-Whitney, p=0.098) and ceca (FIG. 27B; Mann-Whitney, p=0.148)of AUT-GI and Control-GI children obtained by pyrosequencing. (FIGS.27C-27D) Box-and-whisker plots displaying the phyla level abundance ofFirmicutes in the ilea (FIG. 27C; Mann-Whitney, p=0.245) and ceca (FIG.27D; Mann-Whitney, p=0.053) of AUT-GI and Control-GI children obtainedby real-time PCR. Copy number values for Firmicutes are normalizedrelative to total bacteria copy numbers. (FIGS. 27E-27F) Box-and-whiskerplots displaying the abundance of Clostridiales from ileal (FIG. 27E;Mann-Whitney, p=0.072) and cecal (FIG. 27F; Mann-Whitney, p=0.098)biopsies from AUT-GI and Control-GI patients obtained by pyrosequencing.*, p<0.05; †, p<0.1 (trend); n.s., not significant.

FIG. 28 shows genus-level distribution of members of the familiesRuminococcaceae and Lachnospiraceae. (FIGS. 28A-28B) Heatmaprepresentation of the individual patient distributions (by patient) ofRuminococcaceae and Lachnospiraceae genus members in ileal (FIG. 28A)and cecal (FIG. 28B) biopsies from AUT-GI (Patients 1-15) and Control-GI(Patients 16-22) patients. *, genus members contributing to the trendtoward increased Firmicutes in AUT-GI children.

FIG. 29 shows graphs depicting increases in inflammatory markers, suchas CIQ, Resistin, CD163, Tweak, IL17F, and nNOS. These inflammatorymarkers can also serve as biomarkers for diagnosis of human AutismSpectrum Disorders, as well as for detecting the presence of or apredisposition to autism or an autism spectrum disorder.

FIG. 30 depicts graphs of Firmicute/Bacteroidete ratios obtained byreal-time PCR for ilea (FIG. 30A; Mann-Whitney, p=0.0006) and ceca (FIG.30B; Mann-Whitney, p=0.022).

FIG. 31 shows levels of Clostridiales members in AUT-GI patientsstratified by timing of GI onset. FIGS. 31A-B show the abundance ofClostridiales from ileal (FIG. 31A) and cecal (FIG. 31B) biopsies fromAUT-GI and Control-GI patients (n=7), with AUT-GI stratified by whetherthe onset of GI symptoms occurred after (n=5) the onset of autismsymptoms (GI-After) or before and at the same time (n=10) as autismsymptoms (GI-Before/Same). [FIG. 31A: AUT (GI-After) vs. AUT(GI-Before/Same), Mann-Whitney, p=0.028; AUT (GI-Before/Same) vs.Control-GI, Mann-Whitney, p=0.015; AUT (GI-After) vs. Control-GI,Mann-Whitney, p=0.935] [FIG. 31B: AUT (GI-After) vs. AUT(GI-Before/Same), Mann-Whitney, p=0.037; AUT (GI-Before/ Same) vs.Control-GI, Mann-Whitney, p=0.019; AUT (GI-After) vs. Control-GI,Mann-Whitney, p=0.935]. FIGS. 31C-D show the cumulative abundance ofLachnospiraceae and Ruminococcaceae from ileal (FIG. 31C) and cecal(FIG. 31D) biopsies from AUT-GI and Control-GI patients (n=7), withAUT-GI stratified by whether the onset of GI symptoms occurred after(n=5) the onset of autism symptoms or before and at the same time (n=10)as autism symptoms [FIG. 31C: AUT (GI-After) vs. AUT (GI-Before/Same),Mann-Whitney, p=0.028; AUT (GI-Before/Same) vs. Control-GI,Mann-Whitney, p=0.015; AUT (GI-After) vs. Control-GI, Mann-Whitney,p=0.808] [FIG. 31D: AUT (GI-After) vs. AUT (GI-Before/Same),Mann-Whitney, p=0.020; AUT (GI-Before/Same) vs. Control-GI,Mann-Whitney, p=0.011; AUT (GI-After) vs. Control-GI, Mann-Whitney,p=0.685]. *, p<0.05; **, p<0.01; n.s., not significant.

FIG. 31E shows the age at GI onset (in months) for AUT-GI and Control-GIpatients, with AUT-GI stratified by whether GI onset occurred after(n=5) the onset of autism symptoms or before and at the same time (n=10)as autism symptoms [FIG. 31E: AUT (GI-After) vs. AUT (GI-Before/Same),Mann-Whitney, tied p=0.007; AUT (GI-Before/Same) vs. Control-GI,Mann-Whitney, tied p=0.757; AUT (GI-After) vs. Control-GI, Mann-Whitney,tied p=0.027]. *, p<0.05; **,p<0.01; n.s., not significant.

FIG. 32 is a schematic representation of enterocyte-mediated digestionof disaccharides and transport of monosaccharides in the smallintestine. Disaccharidases (SI, MGAM, and LCT) in the enterocyte brushborder break down disaccharides into their component monosaccharides.The monosaccharides, glucose and galactose, are transported from thesmall intestinal lumen into enterocytes by the sodium-dependenttransporter SGLT1. On the basolateral enterocyte membrane, GLUT2,transports glucose, galactose and fructose out of enterocytes and intothe circulation. The expression levels of disaccharidases and hexosetransporters can be controlled, in part, by the transcription factorCDX2.

FIG. 33 is a bar grah showing CDX2 mRNA expression in AUT-GI childrenstratified by number of total disaccharidase and transporterdeficiencies [All 5 deficient (n=10) or fewer than 5 deficient (n=5)]compared to all Control-GI children (n=7). AUT (All 5) vs AUT (<5);Mann-Whitney, p =0.037. AUT (All 5) vs Control; Mann-Whitney, p=0.064.*, p<0.05; **,p<0.01; ***, p<0.001; †, p<0.1 (trend).

FIG. 34 shows the percent difference in abundance of Bacteroidetes,Firmicutes, and Proteobacteria in individual AUT-GI patients. (FIGS.34A-B show bar graphs indicating the percent difference in phylotypeabundance for Bacteroidetes, Firmicutes and Proteobacteria in AUT-Glpatients (#1-15) relative to the Control-GI mean abundance for each ofthe three phylotypes obtained by pyrosequencing of ieal (FIG. 34A) andcecal (FIG. 34B) biopsies.

FIG. 35 shows graphs of increased Betaproteobacteria in AUT-GI childrenis associated with total deficiencies in disaccharidases and hexosetransporters and CDX2 mRNA expression. FIGS. 35A-B show the abundance ofBetaproteobacteria in AUT-GI children with deficiency in all 5disaccharidases and transporters (All 5; n=10), AUT-GI children withdeficiency in fewer than 5 disaccharidases and transporters (<5; n=5),and Control-GI children (n=7) in ileum (FIG. 35A) and cecum (FIG. 35B).(FIG. 35A) Ileum: AUT-GI (All 5) vs. AUT-GI (<5), Mann-Whitney, p=0.028;AUT-GI (All 5) vs. Control-GI, Mann-Whitney, p=0.015; AUT-GI (<5) vs.Control-Gr, Mann-Whitney, p=0.935. (FIG. 35B) Cecum: AUT-GI (All 5) vs.AUT-GI (<5), Mann-Whitney, p=0.014; AUT-GI (All 5) vs. Control-GI,Mann-Whitney, p=0.006; AUT-GI (<5) vs. Control-GI, Mann-Whitney,p=0.808. FIGS. 35C-D show Ileal CDX2 mRNA expression in AUT-GI childrenwith Betaproteobacteria above the 75^(th) percentile of Control-GIchildren [AUT (+) β-proteol.], AUT-GI children with Betaproteobacterialevels below the 75^(th) percentile of Control-GI children [AUT (−)β-proteol.], and Control-GI children in ileum (FIG. 35C) and cecum (FIG.35D). (FIG. 35C) Ileum: AUT (+) β-proteo. (n=8) vs. AUT (−) β-proteo.(n=7), Mann-Whitney, p=0.037; AUT (+) β-proteo. vs. Control-GI (n=7),Mann-Whitney, p=0.064; AUT (−) β-proteo. vs. Control-GI, Mann-Whitney,p=0.749. (FIG. 35D) Cecum: AUT (+) β-proteo. (n=10) vs. AUT (−)β-proteo. (n=5), Mann-Whitney, p=0.028; AUT (+) β-proteo. vs. Control-GI(n=7), Mann-Whitney, p=0.097; AUT (−) β-proteo. vs. Control-GI,Mann-Whitney, p=0.808. *, p<0.05; **,p<0.01; †, p<0.1 (trend); n.s., notsignificant.

FIG. 36 shows the distribution of Sutterella sequences by individualpatient as a percentage of total bacteria 16S rRNA reads from cecalbiopsies from AUT-GI (patients #1-15) and Control-GI (patients #16-22)patients. *, p<0.05.

FIG. 37 is a pie chart indicating the percentage of Sutterella sequencesin the dominant OTU (either OTU 1 or OTU 2) relative to sequences fromsubdominant Sutterella OTUs in ileum and cecum of the sevenSutterella-positive patients. The percentage of the dominant OTU isshown per individual patient.

FIG. 38 is a schematic representation showing the location of PCRprimers and products evaluated in Sutterella-specific PCR assays.

FIG. 39 are photographic images of gels showing PCR-based detection ofSutterella 16S rRNA gene sequences (V6-V8 region and C4-V8 region) inbiopsies from AUT-GI and Control-GI patients. FIG. 39A shows agarose geldetection of 260 by Sutterella products in ileal (4 biopsies/patient)and cecal (4 biopsies/patient) biopsy DNA using SuttFor and SuttRevprimers (V6-V8 region) in conventional PCR assays. FIG. 39B showsagarose gel detection of 715 by Sutterella products in ileal and cecalbiopsy DNA using pan-bacterial primer 515For and SuttRev primer (C4-V8)in conventional PCR assays. Negative control is PCR reagents with watersubstituted for DNA. Positive control is DNA isolated from cultured S.wadsworthensis (ATCC, #51579).

FIG. 40 is a graph that shows quantitation of Sutterella sequences inileal and cecal biopsies from AUT-GI and Control-GI patients using anovel Sutterella-specific real-time PCR assay. Bars in graph show meancopy number in 4 biopsies from ileum (blue) and 4 biopsies from cecum(red)+the standard error mean for each individual patient.

FIG. 41 shows pie charts of the distribution of Sutterella species inileal and cecal biopsies of AUT-GI patients based on C4-V8 products. Theclosest sequence match to known Sutterella isolates was determined usingthe RDP seqmatch tool. The frequency of Sutterella species matches inileal and cecal clone libraries are shown as pie charts for patient #1(FIG. 41A), patient #3 (FIG. 41B), patient #5 (FIG. 41C), patient #7(FIG. 41D), patient #10 (FIG. 41E), patient #11 (FIG. 41F), patient #12(FIG. 41G), patient #24a (FIG. 41H), patient #25a (FIG. 411), patient#27a (FIG. 41J), patient #28a (FIG. 41K), patient #29a (FIG. 41L). *,Note: Sutterella 16S sequences obtained from patient #28a were less than97% similar to the 16S sequence of all known isolates of Sutterellaspecies.

FIG. 42 is a schematic of a phylogenetic tree based on predominant 16SrRNA gene sequences obtained by C4-V8 Sutterella PCR from AUT-GIpatients, Sutterella species isolates, and related species. The tree wasconstructed using the Neighbor-joining method. Bootstrap values (>60%)based on 1000 replicates are shown next to the branches. There were atotal of 653 positions in the final dataset. The evolutionary distanceswere computed using the Jukes-Cantor method and are in the units of thenumber of base substitutions per site. The optimal tree with sum ofbranch length=0.66371685 is shown. The tree is rooted to the outgroupEscherichia coli. Accession numbers are shown in parentheses. AUT-GIpatient sequences are boxed in red.

FIG. 43 shows western immunoblot analysis of AUT-GI and Control-GIpatients' plasma antibody immunoreactivity against S. wadsworthensisantigens. FIG. 43A depicts patients' plasma IgG antibodyimmunoreactivity against S. wadsworthensis antigens: FIG. 43B depictspatients' IgM antibody immunoreactivity against S. wadsworthensisantigens. 2°=Secondary antibody control.

FIG. 44 shows graphs of the abundance distribution of all genus levelclassifications of sequences from pyrosequencing for patients #1, 3, 5and 7. Bar graph showing all ileal genera, in order of highest abundance(top) to lowest abundance (bottom), from (FIG. 44A) patient #1 (32 totalgenera), (FIG. 44B) patient #3 (35 total genera), (FIG. 44C) patient #5(39 total genera), and (FIG. 44D) patient #7 (39 total genera). Theabundances of Sutterella sequences are indicated in red. Noteunclassified family members can represent more than one genus (i.e.Unclassified Lachnospiraceae).

FIG. 45 shows graphs of the abundance distribution of all genus levelclassifications of sequences from pyrosequencing for patients #10, 11,and 12. Bar graph showing all ileal genera, in order of highestabundance (top) to lowest abundance (bottom) from (FIG. 45A) patient #10(32 total genera), (FIG. 45B) patient #11 (39 total genera), and (FIG.45C) patient #12 (44 total genera). The abundances of Sutterellasequences are indicated in red. Note unclassified family members canrepresent more than one genus (i.e. Unclassified Lachnospiraceae).

FIG. 46 depicts Sutterella OTU analysis. Heatmap generated from OTUanalysis of all Sutterella sequences by patient. Note patients #1, 3,10, 11, and 12 cluster together and the majority of Sutterella sequencesare present in OTU 2. Patients #5 and 7 cluster together and themajority of Sutterella sequences are present in OTU 1. Heatmap scalerepresents OTU abundance (expressed as % of total bacterialpyrosequencing reads per patient).

FIG. 47 is a schematic of a phylogenetic tree based on therepresentative 16S rRNA gene sequences obtained by V2 regionpyrosequencing (OTU 1 and OTU 2) from AUT-GI patients, Sutterellaspecies isolates, and related species. The tree was constructed usingthe Neighbor-joining method. Bootstrap values based on 1000 replicatesare shown next to the branches (% bootstrap support). There were a totalof 218 positions in the final dataset. The evolutionary distances werecomputed using the Jukes-Cantor method and are in the units of thenumber of base substitutions per site. The optimal tree with sum ofbranch length=1.01142743 is shown. The tree is rooted to the outgroupEscherichia coli. Accession numbers are shown in parentheses. Thelocation of AUT-GI patients' representative OTU 1 and OTU 2 sequencesare boxed in red.

FIG. 48 is a schematic of a phylogenetic tree based on predominant 16SrRNA gene sequences obtained by V6-V8 Sutterella PCR from AUT-GIpatients, Sutterella species isolates, and related species. The tree wasconstructed using the Neighbor-joining method. Bootstrap values based on1000 replicates are shown next to the branches (% bootstrap support).There were a total of 215 positions in the final dataset. Theevolutionary distances were computed using the Jukes-Cantor method andare in the units of the number of base substitutions per site. Theoptimal tree with sum of branch length=0.67013793 is shown. The tree isrooted to the outgroup Escherichia coli. Accession numbers are shown inparentheses. The location of AUT-GI patients' sequences are boxed inred.

FIG. 49 shows a Sutterella sequence alignment. Clustal W alignment ofthe most abundant Sutterella 16S rRNA gene (C4-V8 region) sequences inthe 12 Sutterella-positive patients. Sequences have had the 515For andSuttRev primer sequences removed. The positions of the beginning(nucleotide position 501) and end (nucleotide position 1176) of thesequences are relative to the 16S rRNA gene of S. wadsworthensis(Accession L37785). Patients 1, 24a (SEQ ID NO: 61); Patients 3, 10, 11,12, 27a, 29a (SEQ ID NO: 62); Patients 5, 7, 25a (SEQ ID NO: 63);Patient 28a (SEQ ID NO: 64).

ABBREVIATIONS used herein: ASD, autism spectrum disorders; GI,gastrointestinal; AUT-GI, children with autistic disorder and GIdisease; Control-GI, normally developing children with GI disease; FA,food allergy; MA, milk-related allergy; WA, wheat-related allergy; AD,atopic disease; SI, sucrase isomaltase; MGAM, maltase glucoamylase; LCT,lactase; SGLT1, sodium-dependent glucose cotransporter; GLUT2, glucosetransporter 2; CDX2, caudal type homeobox 2; OTU, operational taxonomicunit.

DETAILED DESCRIPTION OF THE INVENTION

Autism, one of the ASDs, is mostly diagnosed clinically using behavioralcriteria because few specific biological markers are known fordiagnosing the disease. Autism is a neuropsychiatric developmentaldisorder characterized by impaired verbal communication, non-verbalcommunication, and reciprocal social interaction. It is alsocharacterized by restricted and stereotyped patterns of interests andactivities, as well as the presence of developmental abnormalities by 3years of age (Bailey et al., (1996) J Child Psychol Psychiatry37(1):89-126). Autism-associated disorders, diseases or pathologies cancomprise any metabolic, immune or systemic disorders; gastrointestinaldisorders; epilepsy; congenital malformations or genetic syndromes;anxiety, depression, or AD/HD; or speech delay and motorin-coordination.

Autism spectrum disorders (ASD) are defined by impairments in verbal andnon-verbal communication, social interactions, and repetitive andstereotyped behaviors (DSM-IV-TR criteria, American PsychiatricAssociation, 2000). In addition to these core deficits, previous reportsindicate that the prevalence of gastrointestinal symptoms ranges widelyin individuals with ASD, from 9 to 91% (Buie et al., 2010). Macroscopicand histological observations in ASD include findings of ileo-coloniclymphoid nodular hyperplasia (LNH), enterocolitis, gastritis andesophagitis (Wakefield et al., 2000; Wakefield et al., 2005; Furlano etal., 2001; Torrente et al., 2002; Horvath et al., 1999). Associatedchanges in intestinal inflammatory parameters include higher densitiesof lymphocyte populations, aberrant cytokine profiles, and deposition ofimmunoglobulin (IgG) and complement Clq on the basolateral enterocytemembrane (Furlano et al., 2001; Ashwood and Wakefield, 2006). Functionaldisturbances include increased intestinal permeability (D'Eufemia etal., 1996), compromised sulphoconjugation of phenolic compounds(O'Reilly and Waring, 1993; Alberti et al., 1999), deficient enzymaticactivity of disaccharidases (Horvath et al., 1999), increasedsecretin-induced pancreatico-biliary secretion (Horvath et al., 1999),and abnormal Clostridia taxa (Finegold et al., 2002; Song et al., 2004;Parracho et al., 2005). Some children placed on exclusion diets ortreated with the antibiotic vancomycin are reported to improve incognitive and social function (Knivsberg et al., 2002; Sandler et al.,2000).

The gastrointestinal tract is exposed to an onslaught of foreignmaterial in the form of food, xenobiotics, and microbes. The intestinalmuco-epithelial layer must maximize nutritional uptake of dietarycomponents while maintaining a barrier to toxins and infectious agents.Although some aspects of these functions are host-encoded, others areacquired through symbiotic relationships with microbial flora. Dietarycarbohydrates enter the intestine as monosaccharides (glucose, fructose,and galactose), disaccharides (lactose, sucrose, maltose), or complexpolysaccharides. Following digestion with salivary and pancreaticamylases, carbohydrates are further digested by disaccharidasesexpressed by absorptive enterocytes in the brush border of the smallintestine and transported as monosaccharides across the intestinalepithelium. However, humans lack the glycoside hydrolases andpolysaccharide lyases necessary for cleavage of glycosidic linkagespresent in plant cell wall polysaccharides, oligosaccharides, storagepolysaccharides, and resistant starches. Intestinal bacteria encodingthese enzymes expand the capacity to extract energy from dietarypolysaccharides (Sonnenburg et al., 2008; Flint et al., 2008). As an endproduct of polysaccharide fermentation, bacteria produce short-chainfatty acids (butyrate, acetate, and propionate) that serve as energysubstrates for colonocytes, modulate colonic pH, regulate colonic cellproliferation and differentiation, and contribute to hepaticgluconeogenesis and cholesterol synthesis (Wong et al., 2006; Jacobs etal., 2009). Indigenous microflora also mediate postnatal development ofthe muco-epithelial layer, provide resistance to potential pathogens,regulate development of intraepithelial lymphocytes and Peyer's patches,influence cytokine production and serum immunoglobulin levels, andpromote systemic lymphoid organogenesis (O'Hara and Shanahan, 2006;Macpherson and Harris, 2004).

The prevalence of autism in the US is about 1 in 91 births and, largelydue to changes in diagnostic practices, services, and public awareness.Autism is growing at the fastest pace of any developmental disability(10-17%) (Fombonne, E. (2003). The prevalence of autism. JAMA 289(1):87-9). Care and treatment of autism costs the U.S. healthcare system$90B annually. Early detection and intervention can result in reducinglife-long costs. In the last 5 years, federal funding for autismresearch rose by 16.1%. The Autism Society is currently lobbyingCongress for $37 million for autism monitoring and studies, another$16.5 million for autism screening and academic research. At present,few tools outside psychiatric evaluation are available for diagnosingautism. While a causative link between GI abnormalities and pathology ofautism has yet to be established, a correlation between the twodisorders is relatively well established. Thus, technologiesfacilitating detection and treatment of abnormal gut flora in autisticpatients has great potential utility for diagnosis and treatment.

The present invention provides the discovery and the identification ofGLUT2 as well SGLT1 as biomarkers for human Autism Spectrum Disorders.The present invention provides for methods to use genes encodingcarbohydrate metabolic enzyme molecules (such as sucrase isomaltase,maltase glucoamylase, and lactase) or carbohydrate transportermolecules, or a combination of the two, and corresponding expressionproducts for the diagnosis, prevention and treatment of autism andautism spectrum disorders.

The methods of the invention are useful in various subjects, such ashumans, including adults, children, and developing human fetuses at theprenatal stage.

The GLUT2 gene locus can comprise all GLUT2 sequences or products in acell or organism, including GLUT2 coding sequences, GLUT2 non-codingsequences (e.g., introns), GLUT2 regulatory sequences controllingtranscription and/or translation (e.g., promoter, enhancer, terminator).

A GLUT2 gene, also known as SLC2A2, encodes the glucose transporter 2isoform. It is an integral plasma membrane glycoprotein of the liver,pancreatic islet beta cells, intestine, and kidney epithelium. GLUT2mediates the bidirectional transport of glucose. In the context of theinvention, the GLUT2 gene also encompasses its variants, analogs andfragments thereof, including alleles thereof (e.g., germline mutations)which are related to susceptibility to autism and/or autism spectrumdisorders.

The SGLT1 gene locus can comprise all SGLT1 sequences or products in acell or organism, including SGLT1 coding sequences, SGLT1 non-codingsequences (e.g., introns), SGLT1 regulatory sequences controllingtranscription and/or translation (e.g., promoter, enhancer, terminator).

A SGLT1 gene, also known as SLC5A1, encodes the sodium/glucoseco-transporter 1. The sodium dependent glucose transporter is anintegral plasma membrane glycoprotein of the intestine. SGLT1 mediatesglucose and galactose uptake from the intestinal lumen. Mutations inthis gene have been associated with glucose-galactose malabsorption. Inthe context of the invention, the SGLT1 gene also encompasses itsvariants, analogs and fragments thereof, including alleles thereof(e.g., germline mutations) which are related to susceptibility to autismand/or autism spectrum disorders.

As used herein, “carbohydrate transport activity” means the ability of apolypeptide to bind a carbohydrate, such as glucose, to a transporterprotein, and subsequently facilitate uptake of the carbohydrate from theserum or extracellular millieu into a cell (e.g., a liver cell, orpancreatic n-cell). Glucose transport activity can be measured asdescribed by Hissin et al., 1982, J Clin. Invest. 70(4): 780-90. In oneembodiment, the carbohydrate transport activity is glucose transportactivity, and the activity can be measured by determining glucosetransport activity as described in Hissin as well as the ability todecrease extracellular or serum glucose levels. Non-limiting examples ofa carbohydrate transporter include GLUT 1, GLUT2, GLUT3, GLUT4, GLUT5,GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, and HMIT (seeScheepers et al., JPEN J Parenter Enteral Nutr. 2004 September-Octtober;28(5):364-71).

A sucrase isomaltase (SI) gene encodes a sucrase-isomaltase protein,which is a glucosidase enzyme, that is expressed in the intestinal brushborder. The encoded protein is synthesized as a precursor protein thatis cleaved by pancreatic proteases into two enzymatic subunits, sucraseand isomaltase. The two subunits heterodimerize to form thesucrose-isomaltase complex, which is essential for the digestion ofdietary carbohydrates including starch, sucrose and isomaltose.Mutations in this gene are the cause of congenital sucrase-isomaltasedeficiency. In the context of the invention, the SI gene alsoencompasses its variants, analogs and fragments thereof, includingalleles thereof (e.g., germline mutations) which are related tosusceptibility to autism and/or autism spectrum disorders.

A maltase glucoamylase (MGAM) gene encodes a maltase-glucoamylaseenzyme. It is localized to the brush border membrane and plays a role inthe final steps of digestion of starch. The protein has two catalyticsites identical to those of sucrase-isomaltase, but the proteins areonly 59% homologous. Both are members of glycosyl hydrolase family 31,which has a variety of substrate specificities. In the context of theinvention, the MGAM gene also encompasses its variants, analogs andfragments thereof, including alleles thereof (e.g., germline mutations)which are related to susceptibility to autism and/or autism spectrumdisorders.

A lactase (LCT) gene encodes a glycosyl hydrolase of family 1. Theprotein is integral to plasma membrane and has both phlorizin hydrolaseactivity and lactase activity.

As used herein, “carbohydrate metabolic enzyme activity” includes“sucrase isomaltase activity”, “maltase glucoamylase activity”, “lactaseactivity”, “sucrase activity”, “maltase activity”, “trehalase activity”,“amylase activity”, “cellulase activity”, “glucosidase activity”,“pullulanase activity”, “galactosidase activity”, “alpha-Mannosidaseacivity”, “glucuronidase activity”, “hyaluronidase activity”,“glycosylase activity”, “fucosidase activity”, “hexosaminidaseactivity”, “iduronidase activity”, or “maltase-glucoamylase activity”.“Sucrase isomaltase activity” means the ability of a polypeptide tocatalyze the hydrolysis of sucrose to fructose and glucose and toenzymatically digest polysaccharides at the alpha 1-6 linkages. Sucraseand isomaltase activities can be measured as described by Dahlqvist, A.(1964) Anal. Biochem. 7,18-25 and and the enzyme assays described byGoda et al., Biochem J. 1988 Feb. 15; 250(1): 41-46. “Maltaseglucoamylase activity” means the ability of a polypeptide toenzymatically digest starch, releasing malstose and free glucose, aswell as to catalyze the hydrolysis of the disaccharide maltose. Maltaseand glucoamylase activities can be measured as described by Dahlqvist A.Specificity of the human intestinal disaccharidases and implications forhereditary disaccharide intolerance. J Clin Invest. 1962;41:463-9;Dahlqvist A. Assay of intestinal disaccharidases. Scand J Clin LabInvest. 1984;44:169-72; and Quezada-Calvillo et al., J. Nutr.137:1725-1733, July 2007. “Lactase activity” means the ability of apolypeptide to hydrolyze lactose to galactose and glucose. Lactaseactivity can be measured as described by Dahlqvist A. Specificity of thehuman intestinal disaccharidases and implications for hereditarydisaccharide intolerance. J Clin Invest. 1962;41:463-9; Dahlqvist A.Assay of intestinal disaccharidases. Scand J Clin Lab Invest.1984;44:169-72; and Quezada-Calvillo et al., J. Nutr. 137:1725-1733,July 2007. “Trehalase activity” means the ability of a polypeptide tocatalyze the conversion of the dissacharide trehalose(a-D-glucopyranosyl-1,1-α-D-glucopyranoside) to glucose.

SEQ ID NO: 1 is the human wild type amino acid sequence correspondingto the GLUT2 enzyme (residues 1-524) having GenBank Accession No.NP_000331: 1MTEDKVTGTL VFTVITAVLG SFQFGYDIGV INAPQQVIIS HYRHVLGVPL DDRKAINNYV 61INSTDELPTI SYSMNPKPTP WAEEETVAAA QLITMLWSLS VSSFAVGGMT ASFFGGWLGD 121TLGRIKAMLV ANILSLVGAL LMGFSKLGPS HILIIAGRSI SGLYCGLISG LVPMYIGEIA 181PTALRGALGT FHQLAIVTGI LISQIIGLEF ILGNYDLWHI LLGLSGVRAI LQSLLLFFCP 241ESPRYLYIKL DEEVKAKQSL KRLRGYDDVT KDINEMRKER EEASSEQKVS IIQLFTNSSY 301RQPILVALML HVAQQFSGIN GIFYYSTSIF QTAGISKPVY ATIGVGAVNM VFTAVSVFLV 361EKAGRRSLFL IGMSGMFVCA IFMSVGLVLL NKFSWMSYVS MIAIFLFVSF FEIGPGPIPW 421FMVAEFFSQG PRPAALAIAA FSNWTCNFIV ALCFQYIADF CGPYVFFLFA GVLLAFTLFT 481FFKVPETKGK SFEEIAAEFQ KKSGSAHRPK AAVEMKFLGA TETVSEQ ID NO: 2 is the human wild type nucleic acid sequence correspondingto the GLUT2 enzyme (bps 1-3439) having GenBank Accession No. NM_000340:1 tctggtttgt aacttatgcc taagggacct gctcccattt tctttcctag tggaacaaag 61gtattgaagc cacaggttgc tgaggcaaag cacttattga ttagattccc atcaatattc 121agctgccgct gagaagatta gacttggact ctcaggtctg ggtagcccaa ctcctccctc 181tccttgctcc tcctcctgca atgcataact aggcctaggc agagctgcga ataaacaggc 241aggagctagt caggtgcatg tgccacactc acacaagacc tggaattgac aggactccca 301actagtacaa tgacagaaga taaggtcact gggaccctgg ttttcactgt catcactgct 361gtgctgggtt ccttccagtt tggatatgac attggtgtga tcaatgcacc tcaacaggta 421ataatatctc actatagaca tgttttgggt gttccactgg atgaccgaaa agctatcaac 481aactatgtta tcaacagtac agatgaactg cccacaatct catactcaat gaacccaaaa 541ccaacccctt gggctgagga agagactgtg gcagctgctc aactaatcac catgctctgg 601tccctgtctg tatccagctt tgcagttggt ggaatgactg catcattctt tggtgggtgg 661cttggggaca cacttggaag aatcaaagcc atgttagtag caaacattct gtcattagtt 721ggagctctct tgatggggtt ttcaaaattg ggaccatctc atatacttat aattgctgga 781agaagcatat caggactata ttgtgggcta atttcaggcc tggttcctat gtatatcggt 841gaaattgctc caaccgctct caggggagca cttggcactt ttcatcagct ggccatcgtc 901acgggcattc ttattagtca gattattggt cttgaattta tcttgggcaa ttatgatctg 961tggcacatcc tgcttggcct gtctggtgtg cgagccatcc ttcagtctct gctactcttt 1021ttctgtccag aaagccccag atacctttac atcaagttag atgaggaagt caaagcaaaa 1081caaagcttga aaagactcag aggatatgat gatgtcacca aagatattaa tgaaatgaga 1141aaagaaagag aagaagcatc gagtgagcag aaagtctcta taattcagct cttcaccaat 1201tccagctacc gacagcctat tctagtggca ctgatgctgc atgtggctca gcaattttcc 1261ggaatcaatg gcatttttta ctactcaacc agcatttttc agacggctgg tatcagcaaa 1321cctgtttatg caaccattgg agttggcgct gtaaacatgg ttttcactgc tgtctctgta 1381ttccttgtgg agaaggcagg gcgacgttct ctctttctaa ttggaatgag tgggatgttt 1441gtttgtgcca tcttcatgtc agtgggactt gtgctgctga ataagttctc ttggatgagt 1501tatgtgagca tgatagccat cttcctcttt gtcagcttct ttgaaattgg gccaggcccg 1561atcccctggt tcatggtggc tgagtttttc agtcaaggac cacgtcctgc tgctttagca 1621atagctgcat tcagcaattg gacctgcaat ttcattgtag ctctgtgttt ccagtacatt 1681gcggacttct gtggacctta tgtgtttttc ctctttgctg gagtgctcct ggcctttacc 1741ctgttcacat tttttaaagt tccagaaacc aaaggaaagt cttttgagga aattgctgca 1801gaattccaaa agaagagtgg ctcagcccac aggccaaaag ctgctgtaga aatgaaattc 1861ctaggagcta cagagactgt gtaaaaaaaa aaccctgctt tttgacatga acagaaacaa 1921taagggaacc gtctgttttt aaatgatgat tccttgagca ttttatatcc acatctttaa 1981gtattgtttt atttttatgt gctctcatca gaaatgtcat caaatattac caaaaaagta 2041tttttttaag ttagagaata tatttttgat ggtaagactg taattaagta aaccaaaaag 2101gctagtttat tttgttacac taaagggcag gtggttctaa tatttttagc tctgttcttt 2161ataacaaggt tcttctaaaa ttgaagagat ttcaacatat cattttttta acacataact 2221agaaacctga ggatgcaaca aatatttata tatttgaata tcattaaatt ggaattttct 2281tacccatata tcttatgtta aaggagatat ggctagtggc aataagttcc atgttaaaat 2341agacaactct tccatttatt gcactcagct tttttcttga gtactagaat ttgtattttg 2401cttaaaattt tacttttgtt ctgtattttc atgtggaatg gattatagag tatactaaaa 2461aatgtctata gagaaaaact ttcatttttg gtaggcttat caaaatcttt cagcactcag 2521aaaagaaaac cattttagtt cctttattta atggccaaat ggtttttgca agatttaaca 2581ctaaaaaggt ttcacctgat catatagcgt gggttatcag ttaacattaa catctattat 2641aaaaccatgt tgattccctt ctggtacaat cctttgagtt atagtttgct ttgcttttta 2701attgaggaca gcctggtttt cacatacact caaacaatca tgagtcagac atttggtata 2761ttacctcaaa ttcctaataa gtttgatcaa atctaatgta agaaaatttg aagtaaagga 2821ttgatcactt tgttaaaaat attttctgaa ttattatgtc tcaaaataag ttgaaaaggt 2881agggtttgag gattcctgag tgtgggcttc tgadacttca taaatgttca gcttcagact 2941tttatcaaaa tccctattta attttcctgg aaagactgat tgttttatgg tgtgttccta 3001acataaaata atcgtctcct ttgacatttc cttctttgtc ttagctgtat acagattcta 3061gccaaactat tctatggcca ttactaacac gcattgtaca ctatctatct gcctttacct 3121acataggcaa attggaaata cacagatgat taaacagact ttagcttaca gtcaatttta 3181caattatgga aatatagttc tgatgggtcc caaaagctta gcagggtgct aacgtatctc 3241taggctgttt tctccaccaa ctggagcact gatcaatcct tcttatgttt gctttaatgt 3301gtattgaaga aaagcacttt ttaaaaagta ctctttaaga gtgaaataat taaaaaccac 3361tgaacatttg ctttgttttc taaagttgtt cacatatatg taatttagca gtccaaagaa 3421caagaaattg tttcttttcSEQ ID NO: 3 is the human wild type amino acid sequence correspondingto the SGLT1 enzyme (residues 1-664) having GenBank Accession No.NP_000334: 1MDSSTWSPKT TAVTRPVETH ELIRNAADIS IIVIYFVVVM AVGLWAMFST NRGTVGGFFL 61AGRSMVWWPI GASLFASNIG SGHFVGLAGT GAASGIAIGG FEWNALVLVV VLGWLFVPIY 121IKAGVVTMPE YLRKRFGGQR IQVYLSLLSL LLYIFTKISA DIFSGAIFIN LALGLNLYLA 181IFLLLAITAL YTITGGLAAV IYTDTLQTVI MLVGSLILTG FAFHEVGGYD AFMEKYMKAI 241PTIVSDGNTT FQEKCYTPRA DSFHIFRDPL TGDLPWPGFI FGMSILTLWY WCTDQVIVQR 301CLSAKNMSHV KGGCILCGYL KLMPMFIMVM PGMISRILYT EKIACVVPSE CEKYCGTKVG 361CTNIAYPTLV VELMPNGLRG LMLSVMLASL MSSLTSIFNS ASTLFTMDIY AKVRKRASEK 421ELMIAGRLFI LVLIGISIAW VPIVQSAQSG QLFDYIQSIT SYLGPPIAAV FLLAIFWKRV 481NEPGAFWGLI LGLLIGISRM ITEFAYGTGS CMEPSNCPTI ICGVHYLYFA IILFAISFIT 541IVVISLLTKP IPDVHLYRLC WSLRNSKEER IDLDAEEENI QEGPKETIEI ETQVPEKKKG 601IFRRAYDLFC GLEQHGAPKM TEEEEKAMKM KMTDTSEKPL WRTVLNVNGI ILVTVAVFCH 661AYFASEQ ID NO: 4 is the human wild type nucleic acid sequence correspondingto the SGLT1 enzyme (bps 1-5061) having GenBank Accession No. NM_000343:1 ccccattcgc aggacagctc ttacctgccg ggccgccgcc ccagccaaca gctcagccgg 61gtgctccttc ctgggctcca cgcccggagc tgcttcctga cggtgcagcc gcaaggcatc 121gcaggggccc cgcgctactg ccctgctccc tcaaagtccc aggtcccctc ccctggtgct 181gatcattaac caggaggccg tataaggagc tagcggccct ggcgagaggg aaggacgcaa 241cgctgccacc atggacagta gcacctggag ccccaagacc accgcggtca cccggcctgt 301tgagacccac gagctcattc gcaatgcagc cgatatctcc atcatcgtta tctacttcgt 361ggtagtgatg gccgtcggac tgtgggctat gttttccacc aatcgtggga ctgttggagg 421cttcttcctg gcaggccgaa gtatggtgtg gtggccgatt ggagcctccc tctttgctag 481taacattgga agtggccact ttgtggggct ggccgggact ggggcagctt caggcatcgc 541cattggaggc tttgaatgga atgccctggt tttggtggtt gtgctgggct ggctgtttgt 601ccccatctat attaaggctg gggtggtgac aatgccagag tacctgagga agcggtttgg 661aggccagcgg atccaggtct acctttccct tctgtccctg ctgctctaca ttttcaccaa 721gatctcggca gacatcttct cgggggccat attcatcaat ctggccttag gcctgaatct 781gtatttagcc atctttctct tattggcaat cactgccctt tacacaatta cagggggcct 841ggcggcggtg atttacacgg acaccttgca gacggtgatc atgctggtgg ggtctttaat 901cctgactggg tttgcttttc acgaagtggg aggctatgac gccttcatgg aaaagtacat 961gaaagccatt ccaaccatag tgtctgatgg caacaccacc tttcaggaaa aatgctacac 1021tccaagggcc gactccttcc acatcttccg agatcccctc acgggagacc tcccatggcc 1081tgggttcatc tttgggatgt ccatccttac cttgtggtac tggtgcacag atcaggtcat 1141tgtgcagcgc tgcctctcag ccaagaatat gtctcacgtg aagggtggct gcatcctgtg 1201tgggtatcta aagctgatgc ccatgttcat catggtgatg ccaggaatga tcagccgcat 1261tctgtacaca gaaaaaattg cctgtgtcgt cccttcagaa tgtgagaaat attgcggtac 1321caaggttggc tgtaccaaca tcgcctatcc aaccttagtg gtggagctca tgcccaatgg 1381actgcgaggc ctgatgctat cagtcatgct ggcctccctc atgagctccc tgacctccat 1441cttcaacagc gccagcaccc tcttcaccat ggacatctac gccaaggtcc gcaagagagc 1501atctgagaaa gagctcatga ttgccggaag gttgtttatc ctggtgctga ttggcatcag 1561catcgcctgg gtgcccattg tgcagtcagc acaaagtggg caactcttcg attacatcca 1621gtccatcacc agttacttgg gaccacccat tgcggctgtc ttcctgcttg ctattttctg 1681gaagagagtc aatgagccag gagccttttg gggactgatc ctaggacttc tgattgggat 1741ttcacgtatg attactgagt ttgcttatgg aaccgggagC tgcatggagc ccagcaactg 1801tcccacgatt atctgtgggg tgcactactt gtactttgcc attatcctct tcgccatttc 1861tttcatcacc atcgtggtca tctccctcct caccaaaccc attccggatg tgcatctcta 1921ccgtctgtgt tggagcctgc gcaacagcaa agaggagcgt attgacctgg atgcggaaga 1981ggagaacatc caagaaggcc ctaaggagac cattgaaata gaaacacaag ttcctgagaa 2041gaaaaaagga atcttcagga gagcctatga cctattttgt gggctagagc agcacggtgc 2101acccaagatg actgaggaag aggagaaagc catgaagatg aagatgacgg acacctctga 2161gaagcctttg tggaggacag tgttgaacgt caatggcatc atcctggtga ccgtggctgt 2221cttttgccat gcatattttg cctgagtcct accttttgct gtagatttac catggctgga 2281ctcttactca ccttccttta gtctcgtcct gtggtgttga agggaaatca gccagttgta 2341aattttgccc aggtggataa atgtgtacat gtgtaattat aggctagctg gaagaaaacc 2401attagtttgc tgttaattta tgcatttgaa gccagtgtga tacagccatc tgtacctact 2461ggagctgcag aagggaagtc cactcagtca catccagaaa aaggcagact aagaatcaga 2521agccatgtga ttgatgtctg acgtgagtct gtctcaggta gattccgggt gtcagtgtgg 2581tttataatcc ttgaatattg ttttagaaac tttggtctcc ctggttcctg ccacttttcc 2641tgtccgtcct cctccccatt ttttttttaa aagaaagctg ttttcccctc atcatatccc 2701tcttgagttt tgcctggact ttccctctca agtgtgtcaa tcaggtaaac tgaggaatgc 2761atggaagctg aggatggagc ttgatgggct ccctgtcctg ggtgtttgct ctctgaagtg 2821gaggcctgag gaaggtagta cttccacaaa agggagggac ccgggcccca gcctcaagct 2881agtgggggag gcagatagcc tgaatccagg ggattttctg ggcttcttaa aatgtccatt 2941gtgagttccc cgtgtttggg attccactca ttttggcatt cacagtgcct ggaatgtctt 3001agattttcag caatgcgtgt tgaataaatg aatgacatag gcatttattt ttaaatcttt 3061gcttgctttt tacatgagcc tggcccttag ttaacctttt cttgtggcta cacaaagtat 3121gctcactggt tactaatgac ttgggatgca tttgtcaaac tgattatatt agttttctag 3181ggatgccata acaaagtagc acagaccaga tggctcaagc agcagacatt tattttctca 3241cagttctaga ggctagaagt tggaggccaa gatgtcagca gggttggttt cttctgaggc 3301ctctctcctt ggttgcagat ggtcatatct cactctgtct tccgtggcct tccttttgtc 3361tgtgtcctaa atctactctt ctgataagga catcagtcat attggaatag gacccaccct 3421aatgtcttca ttttaatcac ctctttaaag cccctacctc caaatacagt cacactgtga 3481gaaactgagg gttaggaagt cagcaagtga gtcttgaaga gatactaaac aaacccacaa 3541cacagataaa gtatgcattt tggagatttc caagccagag tctcccgtga aaaaggtaaa 3601cggaagcagt tattgtgcag caaaaggaaa aagaattaca aactgaacgt atgtaggtga 3661ggcaaggcag ggtagggcag ggcctttggg taggctgatc agagggtttt tcaacaataa 3721atcaatggga atgcatttgt tgctcccagg accctggcac cttgactctg gtactatagc 3781atgtcagcaa atacaagcaa agcccaacac tctgatttgc atttatgcca atctaaacta 3841tccggtgttt agtttgattt tttgagtgca ggttcattca aggaccaggt tcccttgtgc 3901tcagggtgaa gtagaaccag aaaacatcgt tatccattcc cagaagtttt ggaagagcct 3961tggtagaaaa gcagaagctg ctttgaccgt gaaaatattt gactcctatc agtttttggt 4021caggagaaga tatccaccta gaccaacctg aggagaaggc tcagagtaca gatatacccc 4081gagcaacgtg atcaatgtcc ttgaaccttc atttttcatc tgaaaacaga gacataaatg 4141cctggctcac agatttaaat gttatacatt gacagcattt atcagtataa catttattta 4201aataagtagg tgctcaatag gtgttggtct tctaacttgt ctacatccca tccccattcc 4261agggtcttca gaattgaagg agagatgttg tatcactgtt agaaggctgc tttgggacat 4321tctgcagcag ggaggaggga ctgtcaaccc ctacaccatg accaccaagt tcctcacctt 4381ggctgagtcc ctaaaactct ctgaacctca ggttcctcca agcataatgc agacttcaca 4441gagctgttgt aaagattagg tgaggtcaat tgatactgct taaaaggccc ggtccgtaga 4501aaatgcccaa taaacattac tgctttcccc ctcaccctac tgcctgaaaa aatattacac 4561ctgtgagact gactttgaga accagtgtgg gtggggagtt gtgcatataa actatttaat 4621gagtaccaaa cacaaaagtc aagcttgtaa aatatcaggc cttgccccag aaagacaaat 4681accacatgat ctcactgata tgtagaatct taaaaagtca aactcagaag cagagagtag 4741aatgatggtt atcaagggct gggggaggga gggactgggg agatgttggt caaatgatac 4801aaaggtttag ttaggtggaa taagttcaga aaatcaattg tacaatgtat caattatagt 4861taatagcaat ataacatata cttgaaaatt gctgagagta gtgtgagtgt tctaccacaa 4921aaaaatatgt gcagtaatag atgttaatta ccttaattta gtcatttcac aatatgtaca 4981tatataaaaa tatgttgtat gccatgagta tatataatta ttatttgtga atttaaaaaa 5041taaaaataat ttccaaaaaa aSEQ ID NO: 5 is the human wild type amino acid sequence correspondingto the sucrase isomaltase (SI) enzyme (residues 1-1827) having GenBankAccession No. NP_001032: 1MARKKFSGLE ISLIVLFVIV TIIAIALIVV LATKTPAVDE ISDSTSTPAT TRVTTNPSDS 61GKCPNVLNDP VNVRINCIPE QFPTEGICAQ RGCCWRPWND SLIPWCFFVD NHGYNVQDMT 121TTSIGVEAKL NRIPSPTLFG NDINSVLFTT QNQTPNRFRF KITDPNNRRY EVPHQYVKEF 181TGPTVSDTLY DVKVAQNPFS IQVIRKSNGK TLFDTSIGPL VYSDQYLQIS TRLPSDYIYG 241IGEQVHKRFR HDLSWKTWPI FTRDQLPGDN NNNLYGHQTF FMCIEDTSGK SFGVFLMNSN 301AMEIFIQPTP IVTYRVTGGI LDFYILLGDT PEQVVQQYQQ LVGLPAMPAY WNLGFQLSRW 361NYKSLDVVKE VVRRNREAGI PFDTQVTDID YMEDKKDFTY DQVAFNGLPQ FVQDLHDHGQ 421KYVIILDPAI SIGRRANGTT YATYERGNTQ HVWINESDGS TPIIGEVWPG LTVYPDFTNP 481NCIDWWANEC SIFHQEVQYD GLWIDMNEVS SFIQGSTKGC NVNKLNYPPF TPDILDKLMY 541SKTICMDAVQ NWGKQYDVHS LYGYSMAIAT EQAVQKVFPN KRSFILTRST FAGSGRHAAH 601WLGDNTASWE QMEWSITGML EFSLFGIPLV GADICGFVAE TTEELCRRWM QLGAFYPFSR 661NHNSDGYEHQ DPAFFGQNSL LVKSSRQYLT IRYTLLPFLY TLFYKAHVFG ETVARPVLHE 721FYEDTNSWIE DTEFLWGPAL LITPVLKQGA DTVSAYIPDA IWYDYESGAK RPWRKQRVDM 781YLPADKIGLH LRGGYIIPIQ EPDVTTTASR KNPLGLIVAL GENNTAKGDF FWDDGETKDT 841IQNGNYILYT FSVSNNTLDI VCTHSSYQEG TTLAFQTVKI LGLTDSVTEV RVAENNQPMN 901AHSNFTYDAS NQVLLIADLK LNLGRNFSVQ WNQIFSENER FNCYPDADLA TEQKCTQRGC 961VWRTGSSLSK APECYFPRQD NSYSVNSARY SSMGITADLQ LNTANARIKL PSDPISTLRV 1021EVKYHKNDML QFKIYDPQKK RYEVPVPLNI PTTPISTYED RLYDVEIKEN PFGIQIRRRS 1081SGRVIWDSWL PGFAFNDQFI QISTRLPSEY IYGFGEVEHT AFKRDLNWNT WGMFTRDQPP 1141GYKLNSYGFH PYYMALEEEG NAHGVFLLNS NAMDVTFQPT PALTYRTVGG ILDFYMFLGP 1201TPEVATKQYH EVIGHPVMPA YWALGFQLCR YGYANTSEVR ELYDAMVAAN IPYDVQYTDI 1261DYMERQLDFT IGEAFQDLPQ FVDKIRGEGM RYIIILDPAI SGNETKTYPA FERGQQNDVF 1321VKWPNTNDIC WAKVWPDLPN ITIDKTLTED EAVNASRAHV AFPDFFRTST AEWWAREIVD 1381FYNEKMKFDG LWIDMNEPSS FVNGTTTNQC RNDELNYPPY FPELTKRTDG LHFRTICMEA 1441EQILSDGTSV LHYDVHNLYG WSQMKPTHDA LQKTTGKRGI VISRSTYPTS GRWGGHWLGD 1501NYARWDNMDK SIIGMMEFSL FGMSYTGADI CGFFNNSEYH LCTRWMQLGA FYPYSRNHNI 1561ANTRRQDPAS WNETFAEMSR NILNIRYTLL PYFYTQMHEI HANGGTVIRP LLHEFFDEKP 1621TWDIFKQFLW GPAFMVTPVL EPYVQTVNAY VPNARWFDYH TGKDIGVRGQ FQTFNASYDT 1681INLHVRGGHI LPCQEPAQNT FYSRQKHMKL IVAADDNQMA QGSLFWDDGE SIDTYERDLY 1741LSVQFNLNQT TLTSTILKRG YINKSETRLG SLHVWGKGTT PVNAVTLTYN GNKNSLPFNE 1801DTTNMILRID LTTHNVTLEE PIEINWSSEQ ID NO: 6 is the human wild type nucleic acid sequence correspondingto the sucrase isomaltase (SI) enzyme (bps 1-6023) having GenBankAccession No. NM_001041: 1ttattttggc agccttatcc aagtctggta caacatagca aagagaacag gctatgaaat 61aagatggcaa gaaagaaatt tagtggattg gaaatctctc tgattgtcct ttttgtcata 121gttactataa tagctattgc cttaattgtt gttttagcaa ctaagacacc tgctgttgat 181gaaattagtg attctacttc aactccagct actactcgtg tgactacaaa tccttctgat 241tcaggaaaat gtccaaatgt gttaaatgat cctgtcaatg tgagaataaa ctgcattcca 301gaacaattcc caacagaggg aatttgtgca cagagaggct gctgctggag gccgtggaat 361gactctctta ttccttggtg cttcttcgtt gataatcatg gttataacgt tcaagacatg 421acaacaacaa gtattggagt tgaagccaaa ttaaacagga taccttcacc tacactattt 481ggaaatgaca tcaacagtgt tctcttcaca actcaaaatc agacacccaa tcgtttccgg 541ttcaagatta ctgatccaaa taatagaaga tatgaagttc ctcatcagta tgtaaaagag 601tttactggac ccacagtttc tgatacgttg tatgatgtga aggttgccca aaacccattt 661agcatccaag ttattaggaa aagcaacggt aaaactttgt ttgacaccag cattggtccc 721ttagtgtact ctgaccagta cttacagatc tcaacccgtc ttccaagtga ttatatttat 781ggtattggag aacaagttca taagagattt cgtcatgatt tatcctggaa aacatggcca 841atttttactc gagaccaact tcctggtgat aataataata atttatacgg ccatcaaaca 901ttctttatgt gtattgaaga tacatctgga aagtcattcg gtgttttttt aatgaatagc 961aatgcaatgg agatttttat ccagcctact ccaatagtaa catatagagt taccggtggc 1021attctggatt tttacatcct tctaggagat acaccagaac aagtagttca acagtatcaa 1081cagcttgttg gactaccagc aatgccagca tattggaatc ttggattcca actaagtcgc 1141tggaattata agtcactaga tgtagtgaaa gaagtggtaa ggagaaaccg ggaagctggc 1201ataccatttg atacacaggt cactgatatt gactacatgg aagacaagaa agactttact 1261tatgatcaag ttgcgtttaa cggactccct caatttgtgc aagatttgca tgaccatgga 1321cagaaatatg tcatcatctt ggaccctgca atttccatag gtcgacgtgc caatggaaca 1381acatatgcaa cctatgagag gggaaacaca caacatgtgt ggataaatga gtcagatgga 1441agtacaccaa ttattggaga ggtatggcca ggattaacag tataccctga tttcactaac 1501ccaaactgca ttgattggtg ggcaaatgaa tgcagtattt tccatcaaga agtgcaatat 1561gatggacttt ggattgacat gaatgaagtt tccagcttta ttcaaggttc aacaaaagga 1621tgtaatgtaa acaaattgaa ttatccaccg tttactcctg atattcttga caaactcatg 1681tattccaaaa caatttgcat ggatgctgtg cagaactggg gtaaacagta tgatgttcat 1741agcctctatg gatacagcat ggctatagcc acagagcaag ctgtacaaaa agtttttcct 1801aataagagaa gcttcattct tacccgctca acatttgctg gatctggaag acatgctgcg 1861cattggttag gagacaatac tgcttcatgg gaacaaatgg aatggtctat aactggaatg 1921ctggagttca gtttgtttgg aatacctttg gttggagcag acatctgtgg atttgtggct 1981gaaaccacag aagaactttg cagaagatgg atgcaacttg gggcatttta tccattttcc 2041agaaaccata attctgacgg atatgaacat caggatcctg cattttttgg gcagaattca 2101cttttggtta aatcatcaag gcagtattta actattcgct acaccttatt acccttcctc 2161tacactctgt tttataaagc ccatgtgttt ggagaaacag tagcaagacc agttcttcat 2221gagttttatg aggatacgaa cagctggatt gaggacactg agtttttgtg gggccctgca 2281ttacttatta ctcctgttct aaaacaggga gcagatactg tgagtgccta catccctgat 2341gctatttggt atgattatga atctggtgca aaaaggccat ggaggaaaca acgggttgat 2401atgtatcttc cagcagacaa aataggatta catcttagag gaggttatat catccccatt 2461caagaaccag atgtaacaac aacagcaagc cgtaagaatc ctctaggact tatagtcgca 2521ttaggtgaaa acaacacagc caaaggagac tttttctggg atgatggaga aactaaagat 2581acaatacaaa atggcaacta catattatat acattttcag tttctaataa cacattagat 2641attgtgtgca cacattcatc atatcaggaa ggaactacct tagcatttca gactgtaaaa 2701atccttgggt tgacagacag tgttacagaa gttagagtgg cggaaaataa tcaaccaatg 2761aacgctcatt ccaatttcac ttatgatgct tctaaccagg ttctcctaat tgcagatctc 2821aaacttaatc ttggaagaaa ctttagtgtt caatggaatc aaattttctc agaaaatgaa 2881agatttaatt gttatccaga tgcagatttg gcaactgaac aaaagtgcac acaacgtggc 2941tgtgtatgga gaacgggttc ttctctatcc aaagcacctg agtgttactt tcccagacaa 3001gataactctt attcagtcaa ctcagctcgc tattcatcca tgggtataac agctgacctc 3061caactaaata ctgcaaatgc cagaataaag ttaccttctg accccatctc aactcttcgt 3121gtggaggtga aatatcacaa aaatgatatg ttgcagttta agatttatga tccccaaaag 3181aagagatatg aagtaccagt accgttaaac attccaacca ccccaataag tacttatgaa 3241gacagacttt atgatgtgga aatcaaggaa aatccttttg gcatccagat tcgacggaga 3301agcagtggaa gagtcatttg ggattcttgg ctgcctggat ttgcttttaa tgaccagttc 3361attcaaatat cgactcgcct gccatcagaa tatatatatg gttttgggga agtggaacat 3421acagcattta agcgagatct gaactggaat acttggggaa tgttcacaag agaccaaccc 3481cctggttaca aacttaattc ctatggattt catccctatt acatggctct ggaagaggag 3541ggcaatgctc atggtgtttt cttactcaac agcaatgcaa tggatgttac attccagcca 3601actcctgctc taacttaccg tacagttgga gggatcttgg atttttatat gtttttgggc 3661ccaactccag aagttgcaac aaagcaatac catgaagtaa ttggccatcc agtcatgcca 3721gcttattggg ctttgggatt ccaattatgt cgttatggat atgcaaatac ttcagaggtt 3781cgggaattat atgacgctat ggtggctgct aacatcccct atgatgttca gtacacagac 3841attgactaca tggaaaggca gctagacttt acaattggtg aagcattcca ggaccttcct 3901cagtttgttg acaaaataag aggagaagga atgagataca ttattatcct ggatccagca 3961atttcaggaa atgaaacaaa gacttaccct gcatttgaaa gaggacagca gaatgatgtc 4021tttgtcaaat ggccaaacac caatgacatt tgttgggcaa aggtttggcc agatttgccc 4081aacataacaa tagataaaac tctaacggaa gatgaagctg ttaatgcttc cagagctcat 4141gtagctttcc cagatttctt caggacttcc acagcagagt ggtgggccag agaaattgtg 4201gacttttaca atgaaaagat gaagtttgat ggtttgtgga ttgatatgaa tgagccatca 4261agttttgtaa atggaacaac tactaatcaa tgcagaaatg acgaactaaa ttatccacct 4321tatttcccag aactcacaaa aagaactgat ggattacatt tcagaacaat ttgcatggaa 4381gctgagcaga ttcttagtga tggaacatca gttttgcatt acgatgttca caatctctat 4441ggatggtcac agatgaaacc tactcatgat gcattgcaga agacaactgg aaaaagaggg 4501attgtaattt ctcgttccac gtatcctact agtggacgat ggggaggaca ctggcttgga 4561gacaactatg cacgatggga caacatggac aaatcaatca ttggtatgat ggaatttagt 4621ctgtttggaa tgtcatatac tggagcagac atctgtggtt ttttcaacaa ctcagaatat 4681catctctgta cccgctggat gcaacttgga gcattttatc catactcaag gaatcacaac 4741attgcaaata ctagaagaca agatcccgct tcctggaatg aaacttttgc tgaaatgtca 4801aggaatattc taaatattag atacacctta ttgccctatt tttacacaca aatgcatgaa 4861attcatgcta atggtggcac tgttatccga ccccttttgc atgagttctt tgatgaaaaa 4921ccaacctggg atatattcaa gcagttctta tggggtccag catttatggt taccccagta 4981ctggaacctt atgttcaaac tgtaaatgcc tacgtcccca atgctcggtg gtttgactac 5041catacaggca aagatattgg cgtcagagga caatttcaaa catttaatgc ttcttatgac 5101acaataaacc tacatgtccg tggtggtcac atcctaccat gtcaagagcc agctcaaaac 5161acattttaca gtcgacaaaa acacatgaag ctcattgttg ctgcagatga taatcagatg 5221gcacagggtt ctctgttttg ggatgatgga gagagtatag acacctatga aagagaccta 5281tatttatctg tacaatttaa tttaaaccag accaccttaa caagcactat attgaagaga 5341ggttacataa ataaaagtga aacgaggctt ggatcccttc atgtatgggg gaaaggaact 5401actcctgtca atgcagttac tctaacgtat aacggaaata aaaattcgct tccttttaat 5461gaagacacta ccaacatgat attacgtatt gatctgacca cacacaatgt tactctagaa 5521gaaccaatag aaatcaactg gtcatgaaga tcaccatcaa ttttagttgt caatgggaaa 5581aaacaccagg atttaagttt cacagcactt acaattttcc ctcttcactt ggttcttgta 5641ctctacaaaa tatagctttc ataacatcga aaagttattt tgtagcgtac atcaatgata 5701atgctaattt tattatagta atgtgacttg gattcaattt taaggcatat ttaacaaaat 5761ttgaatagcc ctatttatcc ttgttaagta tcagctacaa ttgtaaacta gttactaaac 5821atgtatgtaa atagctaaga tataatttaa acgtgatttt taaattaaat aaaattttta 5881tgtaattata tatactatat ttttctcaat gtttagcaga tttaagatat gtaacaacaa 5941ttatttgaag atttaattac ttcttagtat gtgcatttaa ttagaaaaag agaataaaaa 6001atgtaagtgt aaaaaaaaaa aaaSEQ ID NO: 7 is the human wild type amino acid sequence corresponding tothe maltase glucoamylase (MGAM) enzyme (residues 1-1857) having GenBankAccession No. NP_004659: 1MARKKLKKFT TLEIVLSVLL LVLFIISIVL IVLLAKESLK STAPDPGTTG TPDPGTTGTP 61DPGTTGTTHA RTTGPPDPGT TGTTPVSAEC PVVNELERIN CIPDQPPTKA TCDQRGCCWN 121PQGAVSVPWC YYSKNHSYHV EGNLVNTNAG FTARLKNLPS SPVFGSNVDN VLLTAEYQTS 181NRFHFKLTDQ TNNRFEVPHE HVQSFSGNAA ASLTYQVEIS RQPFSIKVTR RSNNRVLFDS 241SIGPLLFADQ FLQLSTRLPS TNVYGLGEHV HQQYRHDMNW KTWPIFNRDT TPNGNGTNLY 301GAQTFFLCLE DASGLSFGVF LMNSNAMEVV LQPAPAITYR TIGGILDFYV FLGNTPEQVV 361QEYLELIGRP ALPSYWALGF HLSRYEYGTL DNMREVVERN RAAQLPYDVQ HADIDYMDER 421RDFTYDSVDF KGFPEFVNEL HNNGQKLVII VDPAISNNSS SSKPYGPYDR GSDMKIWVNS 481SDGVTPLIGE VWPGQTVFPD YTNPNCAVWW TKEFELFHNQ VEFDGIWIDM NEVSNFVDGS 541VSGCSTNNLN NPPFTPRILD GYLFCKTLCM DAVQHWGKQY DIHNLYGYSM AVATAEAAKT 601VFPNKRSFIL TRSTFAGSGK FAAHWLGDNT ATWDDLRWSI PGVLEFNLFG IPMVGPDICG 661FALDTPEELC RRWMQLGAFY PFSRNHNGQG YKDQDPASFG ADSLLLNSSR HYLNIRYTLL 721PYLYTLFFRA HSRGDTVARP LLHEFYEDNS TWDVHQQFLW GPGLLITPVL DEGAEKVMAY 781VPDAVWYDYE TGSQVRWRKQ KVEMELPGDK IGLHLRGGYI FPTQQPNTTT LASRKNPLGL 841IIALDENKEA KGELFWDNGE TKDTVANKVY LLCEFSVTQN RLEVNISQST YKDPNNLAFN 901EIKILGTEEP SNVTVKHNGV PSQTSPTVTY DSNLKVAIIT DIDLLLGEAY TVEWSIKIRD 961EEKIDCYPDE NGASAENCTA RGCIWEASNS SGVPFCYFVN DLYSVSDVQY NSHGATADIS 1021LKSSVYANAF PSTPVNPLRL DVTYHKNEML QFKIYDPNKN RYEVPVPLNI PSMPSSTPEG 1081QLYDVLIKKN PFGIEIRRKS TGTIIWDSQL LGFTFSDMFI RISTRLPSKY LYGFGETEHR 1141SYRRDLEWHT WGMFSRDQPP GYKKNSYGVH PYYMGLEEDG SAHGVLLLNS NAMDVTFQPL 1201PALTYRTTGG VLDFYVFLGP TPELVTQQYT ELIGRPVMVP YWSLGFQLCR YGYQNDSEIA 1261SLYDEMVAAQ IPYDVQYSDI DYMERQLDFT LSPKFAGFPA LINRMKADGM RVILILDPAI 1321SGNETQPYPA FTRGVEDDVF IKYPNDGDIV WGKVWPDFPD VVVNGSLDWD SQVELYRAYV 1381AFPDFFRNST AKWWKREIEE LYNNPQNPER SLKFDGMWID MNEPSSFVNG AVSPGCRDAS 1441LNHPPYMPHL ESRDRGLSSK TLCMESQQIL PDGSLVQHYN VHNLYGWSQT RPTYEAVQEV 1501TGQRGVVITR STFPSSGRWA GHWLGDNTAA WDQLKKSIIG MMEFSLFGIS YTGADICGFF 1561QDAEYEMCVR WMQLGAFYPF SRNHNTJGTR RQDPVSWDVA FVNISRTVLQ TRYTLLPYLY 1621TLMHKAHTEG VTVVRPLLHE FVSDQVTWDI DSQFLLGPAF LVSPVLERNA RNVTAYFPRA 1681RWYDYYTGVD INARGEWKTL PAPLDHINLH VRGGYILPWQ EPALNTHLSR QKFMGFKIAL 1741DDEGTAGGWL FWDDGQSIDT YGKGLYYLAS FSASQNTMQS HIIFNNYITG TNPLKLGYIE 1801IWGVGSVPVT SVSISVSGMV ITPSFNNDPT TQVLSIDVTD RNISLHNFTS LTWISTLSEQ ID NO: 8 is the human wild type nucleic acid sequence correspondingto the maltase glucoamylase (MGAM) enzyme (bps 1-6513) having GenBankAccession No. NM_004668: 1attgctaagc catccttcag acagagaggg agcggctgca agaggtaatg agagatggca 61agaaagaagc tgaaaaaatt tactactttg gagattgtgc tcagtgttct tctgcttgtg 121ttgtttatca tcagtattgt tctaattgtg cttttagcca aagagtcact gaaatcaaca 181gccccagatc ctgggacaac tggtacccca gatcctggga caactggtac cccagatcct 241ggaacaactg gtaccacaca tgctaggaca acgggtcccc cagatcctgg aacaactggt 301accactcctg tttctgctga atgtccagtg gtaaatgaat tggaacgaat taattgcatc 361cctgaccagc cgccaacaaa ggccacatgt gaccaacgtg gctgttgctg gaatccccag 421ggagctgtaa gtgttccctg gtgctactat tccaagaatc atagctacca tgtagagggc 481aaccttgtca acacaaatgc aggattcaca gcccggttga aaaatctgcc ttcttcacca 541gtgtttggaa gcaatgttga caatgttctt ctcacagcag aatatcagac atctaatcgt 601ttccacttta agttgactga ccaaaccaat aacaggtttg aagtgcccca cgaacacgtg 661cagtccttca gtggaaatgc tgctgcttct ttgacctacc aagttgaaat ctccagacag 721ccatttagca tcaaagtgac cagaagaagc aacaatcgtg ttttgtttga ctcgagcatt 781gggcccctac tgtttgctga ccagttcttg cagctctcca ctcgactgcc tagcactaac 841gtgtatggcc tgggagagca tgtgcaccag cagtatcggc atgatatgaa ttggaagacc 901tggcccatat ttaacagaga cacaactccc aatggaaacg gaactaattt gtatggtgcg 961cagacattct tcttgtgcct tgaagatgct agtggattgt cctttggggt gtttctgatg 1021aacagcaatg ccatggaggt tgtccttcag cctgcgccag ccatcactta ccgcaccatt 1081gggggcattc tcgacttcta tgtgttcttg ggaaacactc cagagcaagt tgttcaagaa 1141tatctagagc tcattgggcg gccagccctt ccctcctact gggcgcttgg atttcacctc 1201agtcgttacg aatatggaac cttagacaac atgagggaag tcgtggagag aaatcgcgca 1261gcacagctcc cttatgatgt tcagcatgct gatattgatt atatggatga gagaagggac 1321ttcacttatg attcagtgga ttttaaaggc ttccctgaat ttgtcaacga gttacacaat 1381aatggacaga agcttgtcat cattgtggat ccagccatct ccaacaactc ttcctcaagt 1441aaaccctatg gcccatatga caggggttca gatatgaaga tatgggtgaa tagttcagat 1501ggagtgactc cactcattgg ggaggtctgg cctggacaaa ctgtgtttcc tgattatacc 1561aatcccaact gtgctgtttg gtggacaaag gaatttgagc tttttcacaa tcaagtagag 1621tttgatggaa tctggattga tatgaatgaa gtctccaact ttgttgatgg ttcggtctca 1681ggatgttcca caaacaacct aaataatccc ccattcactc ccagaatcct ggatgggtac 1741ctgttctgca agactctctg tatggatgca gtgcagcact ggggcaagca gtatgacatt 1801cacaatctgt atggctactc catggcggtc gccacagcag aagctgccaa gactgtgttc 1861cctaataaga gaagcttcat tctgacccgt tctacctttg cgggctctgg caagtttgca 1921gcacattggt taggagacaa cactgccacc tgggatgacc tgagatggtc catccctggc 1981gtgcttgagt tcaacctttt tggcatccca atggtgggtc ctgacatatg tggctttgct 2041ttggacaccc ctgaggagct ctgtaggcgg tggatgcagt tgggtgcatt ttatccgttt 2101tctagaaatc acaatggcca aggctacaag gaccaggatc ctgcctcctt tggagctgac 2161tccctgctgt tgaattcctc caggcactac cttaacatcc gctatactct attgccctac 2221ctatacaccc tcttcttccg tgctcacagc cgaggggaca cggtggccag gccccttttg 2281catgagttct acgaggacaa cagcacttgg gatgtgcacc aacagttctt atgggggccc 2341ggcctcctca tcactccagt tctggatgaa ggtgcagaga aagtgatggc atatgtgcct 2401gatgctgtct ggtatgacta cgagactggg agccaagtga gatggaggaa gcaaaaagtc 2461gagatggaac ttcctggaga caaaattgga cttcaccttc gaggaggcta catcttcccc 2521acacagcagc caaatacaac cactctggcc agtcgaaaga accctcttgg tcttatcatt 2581gccctagatg agaacaaaga agcaaaagga gaacttttct gggataatgg ggaaacgaag 2641gatactgtgg ccaataaagt gtatctttta tgtgagtttt ctgtcactca aaaccgcttg 2701gaggtgaata tttcacaatc aacctacaag gaccccaata atttagcatt taatgagatt 2761aaaattcttg ggacggagga acctagcaat gttacagtga aacacaatgg tgtcccaagt 2821cagacttctc ctacagtcac ttatgattct aacctgaagg ttgccattat cacagatatt 2881gatcttctcc tgggagaagc atacacagtg gaatggagca taaagataag ggatgaagaa 2941aaaatagact gttaccctga tgagaatggt gcttctgccg aaaactgcac tgcccgtggc 3001tgtatctggg aggcatccaa ttcttctgga gtcccttttt gctattttgt caacgaccta 3061tactctgtca gtgatgttca gtataattcc catggggcca cagctgacat ctccttaaag 3121tcttccgttt atgccaatgc cttcccctcc acacccgtga acccccttcg cctggatgtc 3181acttaccata agaatgaaat gctgcagttc aagatttatg atcccaacaa gaatcggtat 3241gaagttccag tccctctgaa catacccagc atgccatcca gcacccctga gggtcaactc 3301tatgatgtgc tcattaagaa gaatccattt gggattgaaa ttcgccggaa gagtacaggc 3361actataattt gggactctca gctccttggc tttaccttca gtgacatgtt tatccgcatc 3421tccacccgcc ttccctccaa gtacctctat ggctttgggg aaactgagca caggtcctat 3481aggagagact tggagtggca cacttggggg atgttctccc gagaccagcc cccagggtac 3541aagaagaatt cctatggtgt ccacccctac tacatggggc tggaggagga cggcagtgcc 3601catggagtgc tcctgctgaa cagcaatgcc atggatgtga cgttccagcc cctgcctgcc 3661ttgacatacc gcaccacagg gggagttctg gacttttatg tgttcttggg gccgactcca 3721gagcttgtca cccagcagta cactgagttg attggccggc ctgtgatggt accttactgg 3781tctttggggt tccagctgtg tcgctatggc taccagaatg actctgagat cgccagcttg 3841tatgatgaga tggtggctgc ccagatccct tatgatgtgc agtactcaga catcgactac 3901atggagcggc agctggactt caccctcagc cccaagtttg ctgggtttcc agctctgatc 3961aatcgcatga aggctgatgg gatgcgggtc atcctcattc tggatccagc catttctggc 4021aatgagacac agccttatcc tgccttcact cggggcgtgg aggatgacgt cttcatcaaa 4081tacccaaatg atggagacat tgtctgggga aaggtctggc ctgattttcc tgatgttgtt 4141gtgaatgggt ctctagactg ggacagccaa gtggagctat atcgagctta tgtggccttc 4201ccagactttt tccgtaattc aactgccaag tggtggaaga gggaaataga agaactatac 4261aacaatccac agaatccaga gaggagcttg aagtttgatg gcatgtggat tgatatgaat 4321gaaccatcaa gcttcgtgaa tggggcagtt tctccaggct gcagggacgc ctctctgaac 4381caccctccct acatgccaca tttggagtcc agggacaggg gcctgagcag caagaccctt 4441tgtatggaga gtcagcagat cctcccagac ggctccctgg tgcagcacta caacgtgcac 4501aacctgtatg ggtggtccca gaccagaccc acatacgaag ccgtgcagga ggtgacggga 4561cagcgagggg tcgtcatcac ccgctccaca tttccctctt ctggccgctg ggcaggacat 4621tggctgggag acaacacggc cgcatgggat cagctgaaga agtctatcat tggcatgatg 4681gagttcagcc tcttcggcat atcctatacg ggagcagata tctgtgggtt ctttcaagat 4741gctgaatatg agatgtgtgt tcgctggatg cagctggggg ccttttaccc cttctcaaga 4801aaccacaaca.ccattgggac caggagacaa gaccctgtgt cctgggatgt tgcttttgtg 4861aatatttcca gaactgtcct gcagaccaga tacaccctgt tgccatatct gtataccttg 4921atgcataagg cccacacgga gggcgtcact gttgtgcggc ctctgctcca tgagtttgtg 4981tcagaccagg tgacatggga catagacagt cagttcctgc tgggcccagc cttcctggtc 5041agccctgtcc tggagcgtaa tgccagaaat gtcactgcat atttccctag agcccgctgg 5101tatgattact acacgggtgt ggatattaat gcaagaggag agtggaagac cttgccagcc 5161cctcttgacc acattaatct tcatgtccgt gggggctaca tcctgccctg gcaagagcct 5221gcactgaaca cccacttaag ccgccagaaa ttcatgggct tcaaaattgc cttggatgat 5281gaaggaactg ctgggggctg gctcttctgg gatgatgggc aaagcattga tacctatggg 5341aaaggactct attacttggc cagcttttct gccagccaga atacgatgca aagccatata 5401attttcaaca attacatcac tggtacaaat cctttgaaac tgggctacat tgaaatctgg 5461ggagtgggca gtgtccccgt taccagtgtc agcatctctg tgagtggcat ggtcataaca 5521ccctccttca acaatgaccc cacgacacag gtattaagca tcgatgtgac tgacagaaac 5581atcagcctac ataattttac ttcattgacg tggataagca ctctgtgaat ttttacagca 5641agattctaac taactatgaa tgactttgaa actacttata cttcatactc ataaaaatta 5701ttgtgtgttg ctaatttgtt catacccact attggtgaaa tatttctgtt aattttgtta 5761tatgtttttt gtgtgaaccc taaaggttaa accttagccc tgtgggatag gcagttaggg 5821aggtgtggaa aatctatgca ttaccttaat gtctctgtgt ggttagtatg gtagtgactg 5881ttcatcatat gacatttact gaagatgaac tgggtccatg atgaagtgtg tgtatgtcca 5941cgtttgtaat catagaatgg accccattct tttgttaaat acacaagaga aagctttctg 6001tgacagttcc aggtcttgaa gctaatcagc atctcaagaa agtatccaga aagaacatct 6061gctagttggt tataggcggt gggaggaata atatacctaa ttggttatag gtggggggag 6121catgataagc aaagaaaagg caaacacaag gaaagatcag atgaaacaga agatgatagt 6181aaaagtgatc ctaagtaaga acataatgta aaattgtcag cagcctcatg gggaggaaaa 6241aggaagagtc aactcacttg aagaagaggg tcttgagaaa tccttagcat aaagggctac 6301tggtgagatt gagatctgag caggcaaagc tcaaaagaga gtttggaggt taaaaataat 6361ttatttttgc agtagtgtgc tttgaaatgt gtaaatctta tttctaatgt atacaaccac 6421atttcacata aaaatatgca atttatatgc cagataaaaa taaaacaagt gaatttgcaa 6481gtgaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaSEQ ID NO: 9 is the human wild type amino acid sequence correspondingto the lactase (LCT) enzyme (residues 1-1927) having GenBank AccessionNo. NP_002290: 1MELSWHVVFI ALLSFSCWGS DWESDRNFIS TAGPLTNDLL HNLSGLLGDQ SSNFVAGDKD 61MYVCHQPLPT FLPEYFSSLH ASQITHYKVF LSWAQLLPAG STQNPDEKTV QCYRRLLKAL 121KTARLQPMVI LHHQTLPAST LRRTEAFADL FADYATFAFH SFGDLVGIWF TFSDLEEVIK 181ELPHQESRAS QLQTLSDAHR KAYEIYHESY AFQGGKLSVV LRAEDIPELL LEPPISALAQ 241DTVDFLSLDL SYECQNEASL RQKLSKLQTI EPKVKVFIFN LKLPDCPSTM KNPASLLFSL 301FEAINKDQVL TIGFDINEFL SCSSSSKKSM SCSLTGSLAL QPDQQQDHET TDSSPASAYQ 361RIWEAFANQS RAERDAFLQD TFPEGFLWGA STGAFNVEGG WAEGGRGVSI WDPRRPLNTT 421EGQATLEVAS DSYHKVASDV ALLCGLRAQV YKFSISWSRI FPMGHGSSPS LPGVAYYNKL 481IDRLQDAGIE PMATLFHWDL PQALQDHGGW QNESVVDAFL DYAAFCFSTF GDRVKLWVTF 541HEPWVMSYAG YGTGQHPPGI SDPGVASFKV AHLVLKAHAR TWHHYNSHHR PQQQGHVGIV 601LNSDWAEPLS PERPEDLRAS ERFLHFMLGW FAHPVFVDGD YPATLRTQIQ QMNRQCSHPV 661AQLPEFTEAE KQLLKGSADF LGLSHYTSRL ISNAPQNTCI PSYDTIGGFS QHVNHVWPQT 721SSSWIRVVPW GIRRLLQFVS LEYTRGKVPI YLAGNGMPIG ESENLFDDSL RVDYFNQYIN 781EVLKAIKEDS VDVRSYIARS LIDGFEGPSG YSQRFGLHHV NFSDSSKSRT PRKSAYFFTS 841IIEKNGFLTK GAKRLLPPNT VNLPSKVRAF TFPSEVPSKA KVVWEKFSSQ PKFERDLFYH 901GTFRDDFLWG VSSSAYQIEG AWDADGKGPS IWDNFTHTPG SNVKDNATGD IACDSYHQLD 961ADLNMLRALK VKAYRFSISW SRIFPTGRNS SINSHGVDYY NRLINGLVAS NIFPMVTLFH 1021WDLPQALQDI GGWENPALID LFDSYADFCF QTFGDRVKFW MTFNEPMYLA WLGYGSGEFP 1081PGVKDPGWAP YRIAHAVIKA HARVYHTYDE KYRQEQKGVI SLSLSTHWAE PKSPGVPRDV 1141EAADRMLQFS LGWFAHPIFR NGDYPDTMKW KVGNRSELQH LATSRLPSFT EEEKRFIRAT 1201ADVFCLNTYY SRIVQHKTPR LNPPSYEDDQ EMAEEEDPSW PSTAMNRAAP WGTRRLLNWI 1261KEEYGDIPIY ITENGVGLTN PNTEDTDRIF YHKTYINEAL KAYRLDGIDL RGYVAWSLMD 1321NFEWLNGYTV KFGLYHVDFN NTNRPRTARA SARYYTEVIT NNGMPLARED EFLYGRFPEG 1381FIWSAASAAY QIEGAWRADG KGLSIWDTFS HTPLRVENDA IGDVACDSYH KIAEDLVTLQ 1441NLGVSHYRFS ISWSRILPDG TTRYINEAGL NYYVRLIDTL LAASIQPQVT IYHWDLPQTL 1501QDVGGWENET IVQRFKEYAD VLFQRLGDKV KFWITLNEPF VIAYQGYGYG TAAPGVSNRP 1561GTAPYIVGHN LIKAHAEAWH LYNDVYRASQ GGVISITISS DWAEPRDPSN QEDVEAARRY 1621VQFMGGWFAH PIFKNGDYNE VMKTRIRDRS LAAGLNKSRL PEFTESEKRR INGTYDFFGF 1681NHYTTVLAYN LNYATAISSF DADRGVASIA DRSWPDSGSF WLKMTPFGFR RILNWLKEEY 1741NDPPIYVTEN GVSQREETDL NDTARIYYLR TYINEALKAV QDKVDLRGYT VWSAMDNFEW 1801ATGFSERFGL HFVNYSDPSL PRIPKASAKF YASVVRCNGF PDPATGPHAC LHQPDAGPTI 1861SPVRQEEVQF LGLMLGTTEA QTALYVLFSL VLLGVCGLAF LSYKYCKRSK QGKTQRSQQE 1921LSPVSSFSEQ ID NO: 10 is the human wild type nucleic acid sequence correspondingto the lactase(LCT) enzyme (bps 1-6274) having GenBank Accession No.NM_002299: 1gttcctagaa aatggagctg tcttggcatg tagtctttat tgccctgcta agtttttcat 61gctgggggtc agactgggag tctgatagaa atttcatttc caccgctggt cctctaacca 121atgacttgct gcacaacctg agtggtctcc tgggagacca gagttctaac tttgtagcag 181gggacaaaga catgtatgtt tgtcaccagc cactgcccac tttcctgcca gaatacttca 241gcagtctcca tgccagtcag atcacccatt ataaggtatt tctgtcatgg gcacagctcc 301tcccagcagg aagcacccag aatccagacg agaaaacagt gcagtgctac cggcgactcc 361tcaaggccct caagactgca cggcttcagc ccatggtcat cctgcaccac cagaccctcc 421ctgccagcac cctccggaga accgaagcct ttgctgacct cttcgccgac tatgccacat 481tcgccttcca ctccttcggg gacctagttg ggatctggtt caccttcagt gacttggagg 541aagtgatcaa ggagcttccc caccaggaat caagagcgtc acaactccag accctcagtg 601atgcccacag aaaagcctat gagatttacc acgaaagcta tgcttttcag ggcggaaaac 661tctctgttgt cctgcgagct gaagatatcc cggagctcct gctagaacca cccatatctg 721cgcttgccca ggacacggtc gatttcctct ctcttgattt gtcttatgaa tgccaaaatg 781aggcaagtct gcggcagaag ctgagtaaat tgcagaccat tgagccaaaa gtgaaagttt 841tcatcttcaa cctaaaactc ccagactgcc cctccaccat gaagaaccca gccagtctgc 901tcttcagcct ttttgaagcc ataaataaag accaagtgct caccattggg tttgatatta 961atgagtttct gagttgttca tcaagttcca agaaaagcat gtcttgttct ctgactggca 1021gcctggccct tcagcctgac cagcagcagg accacgagac cacggactcc tctcctgcct 1081ctgcctatca gagaatctgg gaagcatttg ccaatcagtc cagggcggaa agggatgcct 1141tcctgcagga tactttccct gaaggcttcc tctggggtgc ctccacagga gcctttaacg 1201tggaaggagg ctgggccgag ggtgggagag gggtgagcat ctgggatcca cgcaggcccc 1261tgaacaccac tgagggccaa gcgacgctgg aggtggccag cgacagttac cacaaggtag 1321cctctgacgt cgccctgctt tgcggcctcc gggctcaggt gtacaagttc tccatctcct 1381ggtcccggat cttccccatg gggcacggga gcagccccag cctcccaggc gttgcctact 1441acaacaagct gattgacagg ctacaggatg cgggcatcga gcccatggcc acgctgttcc 1501actgggacct gcctcaggcc ctgcaggatc atggtggatg gcagaatgag agcgtggtgg 1561atgccttcct ggactatgcg gccttctgct tctccacatt tggggaccgt gtgaagctgt 1621gggtgacctt ccatgagccg tgggtgatga gctacgcagg ctatggcacc ggccagcacc 1681ctcccggcat ctctgaccca ggagtggcct cttttaaggt ggctcacttg gtcctcaagg 1741ctcatgccag aacttggcac cactacaaca gccatcatcg cccacagcag caggggcacg 1801tgggcattgt gctgaactca gactgggcag aacccctgtc tccagagagg cctgaggacc 1861tgagagcctc tgagcgcttc ttgcacttca tgctgggctg gtttgcacac cccgtctttg 1921tggatggaga ctacccagcc accctgagga cccagatcca acagatgaac agacagtgct 1981cccatcctgt ggctcaactc cccgagttca cagaggcaga gaagcagctc ctgaaaggct 2041ctgctgattt tctgggtctg tcgcattaca cctcccgcct catcagcaac gccccacaaa 2101acacctgcat ccctagctat gataccattg gaggcttctc ccaacacgtg aaccatgtgt 2161ggccccagac ctcatcctct tggattcgtg tggtgccctg ggggataagg aggctgttgc 2221agtttgtatc cctggaatac acaagaggaa aagttccaat ataccttgcc gggaatggca 2281tgcccatagg ggaaagtgaa aatctctttg atgattcctt aagagtagac tacttcaatc 2341aatatatcaa tgaggtgctc aaggctatca aggaagactc tgtggatgtt cgttcctaca 2401ttgctcgttc cctcattgat ggcttcgaag gcccttctgg ttacagccag cggtttggcc 2461tgcaccacgt caacttcagc gacagcagca agtcaaggac tcccaggaaa tctgcctact 2521ttttcactag catcatagaa aagaacggtt tcctcaccaa gggggcaaaa agactgctac 2581cacctaatac agtaaacctc ccctccaaag tcagagcctt cacttttcca tctgaggtgc 2641cctccaaggc taaagtcgtt tgggaaaagt tctccagcca acccaagttc gaaagagatt 2701tgttctacca cgggacgttt cgggatgact ttctgtgggg cgtgtcctct tccgcttatc 2761agattgaagg cgcgtgggat gccgatggca aaggccccag catctgggat aactttaccc 2821acacaccagg gagcaatgtg aaagacaatg ccactggaga catcgcctgt gacagctatc 2881accagctgga tgccgatctg aatatgctcc gagctttgaa ggtgaaggcc taccgcttct 2941ctatctcctg gtctcggatt ttcccaactg ggagaaacag ctctatcaac agtcatgggg 3001ttgattatta caacaggctg atcaatggct tggtggcaag caacatcttt cccatggtga 3061cattgttcca ttgggacctg ccccaggccc tccaggatat cggaggctgg gagaatcctg 3121ccttgattga cttgtttgac agctacgcag acttttgttt ccagaccttt ggtgatagag 3181tcaagttttg gatgactttt aatgagccca tgtacctggc atggctaggt tatggctcag 3241gggaatttcc cccaggggtg aaggacccag gctgggcacc atataggata gcccacgccg 3301tcatcaaagc ccatgccaga gtctatcaca cgtacgatga gaaatacagg caggagcaga 3361agggggtcat ctcgctgagc ctcagtacac actgggcaga gcccaagtca ccaggggtcc 3421ccagagatgt ggaagccgct gaccgaatgc tgcagttctc cctgggctgg tttgctcacc 3481ccatttttag aaacggagac tatcctgaca ccatgaagtg gaaagtgggg aacaggagtg 3541aactgcagca cttagccacc tcccgcctgc caagcttcac tgaggaagag aagaggttca 3601tcagggcgac ggccgacgtc ttctgcctca acacgtacta ctccagaatc gtgcagcaca 3661aaacacccag gctaaaccca ccctcctacg aagacgacca ggagatggct gaggaggagg 3721acccttcgtg gccttccacg gcaatgaaca gagctgcgcc ctgggggacg cgaaggctgc 3781tgaactggat caaggaagag tatggtgaca tccccattta catcaccgaa aacggagtgg 3841ggctgaccaa tccgaacacg gaggatactg ataggatatt ttaccacaaa acctacatca 3901atgaggcttt gaaagcctac aggctcgatg gtatagacct tcgagggtat gtcgcctggt 3961ctctgatgga caactttgag tggctaaatg gctacacggt caagtttgga ctgtaccatg 4021ttgatttcaa caacacgaac aggcctcgca cagcaagagc ctccgccagg tactacacag 4081aggtcattac caacaacggc atgccactgg ccagggagga tgagtttctg tacggacggt 4141ttcctgaggg cttcatctgg agtgcagctt ctgctgcata tcagattgaa ggtgcgtgga 4201gagcagatgg caaaggactc agcatttggg acacgttttc tcacacacca ctgagggttg 4261agaacgatgc cattggagac gtggcctgtg acagttatca caagattgct gaggatctgg 4321tcaccctgca gaacctgggc gtgtcccact accgtttttc catctcctgg tctcgcatcc 4381tccctgatgg aaccaccagg tacatcaatg aagcgggcct gaactactac gtgaggctca 4441tcgatacact gctggccgcc agcatccagc cccaggtgac catttaccac tgggacctac 4501cacagacgct ccaagatgta ggaggctggg agaatgagac catcgtgcag cggtttaagg 4561agtatgcaga tgtgctcttc cagaggctgg gagacaaggt gaagttttgg atcacgctga 4621atgagccctt tgtcattgct taccagggct atggctacgg aacagcagct ccaggagtct 4681ccaataggcc tggcactgcc ccctacattg ttggccacaa tctaataaag gctcatgctg 4741aggcctggca tctgtacaac gatgtgtacc gcgccagtca aggtggcgtg atttccatca 4801ccatcagcag tgactgggct gaacccagag atccctctaa ccaggaggat gtggaggcag 4861ccaggagata tgttcagttc atgggaggct ggtttgcaca tcctattttc aagaatggag 4921attacaatga ggtgatgaag acgcggatcc gtgacaggag cttggctgca ggcctcaaca 4981agtctcggct gccagaattt acagagagtg agaagaggag gatcaacggc acctatgact 5041tttttgggtt caatcactac accactgtcc tcgcctacaa cctcaactat gccactgcca 5101tctcttcttt tgatgcagac agaggagttg cttccatcgc agatcgctcg tggccagact 5161ctggctcctt ctggctgaag atgacgcctt ttggcttcag gaggatcctg aactggttaa 5221aggaggaata caatgaccct ccaatttatg tcacagagaa tggagtgtcc cagcgggaag 5281aaacagacct caatgacact gcaaggatct actaccttcg gacttacatc aatgaggccc 5341tcaaagctgt gcaggacaag gtggaccttc gaggatacac agtttggagt gcgatggaca 5401attttgagtg ggccacaggc ttttcagaga gatttggtct gcattttgtg aactacagtg 5461acccttctct gccaaggatc cccaaagcat cagcgaagtt ctacgcctct gtggtccgat 5521gcaatggctt ccctgacccc gctacagggc ctcacgcttg tctccaccag ccagatgctg 5581gacccaccat cagccccgtg agacaggagg aggtgcagtt cctggggcta atgctcggca 5641ccacagaagc acagacagct ttgtacgttc tcttttctct tgtgcttctt ggagtctgtg 5701gcttggcatt tctgtcatac aagtactgca agcgctctaa gcaagggaaa acacaacgaa 5761gccaacagga attgagcccg gtgtcttcat tctgatgagt taccacctca agttctatga 5821agcaggccta gtttcttcat ctatgtttac cggccaccaa acaccttagg gtcttagact 5881ctgctgatac tggacttctc cataaagtcc tgctgcaccg ttagagatga ctttaatctt 5941gaatgatttc gacttgctga gtaaaatgga aatatctcca tcttgctcca gtatcagagt 6001tcatttgggc atttgagaag caagtagctc ttgcggaaac gtgtagatac tggtctagtg 6061ggtctgtgaa ccacttaatt gaacttaaca gggctgtttt aagtttcaga gttgttaagg 6121gttgttaagg gagcaaaaac cgtaaaaatc cttcctataa gaagaaatca actccattgc 6181atagactgca atatcatctc ctgcccttct gcaagctctc cctagcttca catcttgtgt 6241tttccagaaa ataaaaacag cagactgtcc tttc

As used herein, a “carbohydrate transporter molecule” means a nucleicacid which encodes a polypeptide that exhibits carbohydrate transporteractivity, or a polypeptide or peptidomimetic that exhibits carbohydratetransporter activity. For example, a carbohydrate transporter moleculecan include the human GLUT2 protein (e.g., having the amino acidsequence shown in SEQ ID NO: 1), or a variant thereof, such as afragment thereof, that exhibits carbohydrate transporter activity. Forexample, a carbohydrate transporter molecule can include the human SGLT1protein (e.g., having the amino acid sequence shown in SEQ ID NO: 3), ora variant thereof, such as a fragment thereof, that exhibitscarbohydrate transporter activity. The nucleic acid can be any type ofnucleic acid, including genomic DNA, complementary DNA (cDNA), syntheticor semi-synthetic DNA, as well as any form of corresponding RNA. Forexample, a carbohydrate transporter molecule can comprise a recombinantnucleic acid encoding human GLUT2 protein or human SGLT1 protein. In oneembodiment, a carbohydrate transporter molecule can comprise anon-naturally occurring nucleic acid created artificially (such as byassembling, cutting, ligating or amplifying sequences). A carbohydratetransporter molecule can be double-stranded. A carbohydrate transportermolecule can be single-stranded. The carbohydrate transporter moleculesof the invention can be obtained from various sources and can beproduced according to various techniques known in the art. For example,a nucleic acid that is a carbohydrate transporter molecule can beobtained by screening DNA libraries, or by amplification from a naturalsource. The carbohydrate transporter molecules of the invention can beproduced via recombinant DNA technology and such recombinant nucleicacids can be prepared by conventional techniques, including chemicalsynthesis, genetic engineering, enzymatic techniques, or a combinationthereof. Non-limiting examples of a carbohydrate transporter molecule,that is a nucleic acid, is the nucleic acid having the nucleotidesequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Another example of acarbohydrate transporter molecule is a fragment of a nucleic acid havingthe sequence shown in SEQ ID NO: 2 or SEQ ID NO:4, wherein the fragmentis exhibits carbohydrate transporter activity.

As used herein, a “carbohydrate metabolic enzyme molecule” means anucleic acid which encodes a polypeptide that exhibits carbohydratemetabolic enzyme activity, or a polypeptide or peptidomimetic thatexhibits carbohydrate metabolic enzyme activity. For example, acarbohydrate metabolic enzyme molecule can include the humansucrase-isomaltase (SI) protein (e.g., having the amino acid sequenceshown in SEQ ID NO: 5), or a variant thereof, such as a fragmentthereof, that exhibits carbohydrate metabolic enzyme activity. Forexample, a carbohydrate metabolic enzyme molecule can include the humanmaltase-glucoamylase protein (e.g., having the amino acid sequence shownin SEQ ID NO: 7), or a variant thereof, such as a fragment thereof, thatexhibits carbohydrate metabolic enzyme activity. For example, acarbohydrate metabolic enzyme molecule can include the human lactaseprotein (e.g., having the amino acid sequence shown in SEQ ID NO: 9), ora variant thereof, such as a fragment thereof, that exhibitscarbohydrate metabolic enzyme activity. The nucleic acid can be any typeof nucleic acid, including genomic DNA, complementary DNA (cDNA),synthetic or semi-synthetic DNA, as well as any form of correspondingRNA. For example, a carbohydrate metabolic enzyme molecule can comprisea recombinant nucleic acid encoding human sucrase-isomaltase (SI)protein, human maltase-glucoamylase protein, or human lactase protein.In one embodiment, a carbohydrate metabolic enzyme molecule can comprisea non-naturally occurring, nucleic acid created artificially (such as byassembling, cutting, ligating or amplifying sequences). A carbohydratemetabolic enzyme molecule can be double-stranded. A carbohydratemetabolic enzyme molecule can be single-stranded. The carbohydratemetabolic enzyme molecules of the invention can be obtained from varioussources and can be produced according to various techniques known in theart. For example, a nucleic acid that is a carbohydrate metabolic enzymemolecule can be obtained by screening DNA libraries, or by amplificationfrom a natural source. The carbohydrate metabolic enzyme molecules ofthe invention can be produced via recombinant DNA technology and suchrecombinant nucleic acids can be prepared by conventional techniques,including chemical synthesis, genetic engineering, enzymatic techniques,or a combination thereof. A non-limiting example of a carbohydratemetabolic enzyme, that is a nucleic acid, is the nucleic acid having thenucleotide sequence shown in SEQ ID NO: 6, 8, or 10. Another example ofa carbohydrate metabolic enzyme molecule is a fragment of a nucleic acidhaving the sequence shown in SEQ ID NO: 6, 8, or 10, wherein thefragment is exhibits carbohydrate metabolic enzyme activity.

According to this invention, a carbohydrate transporter moleculeencompass es orthologs of human GLUT2 and SGLT1. According to thisinvention, a carbohydrate metabolic enzyme molecule encompass orthologsof human sucrase-isomaltase (SI), human maltase-glucoamylase, and humanlactase. For example, a carbohydrate transporter molecule or acarbohydrate metabolic enzyme molecule encompass the orthologs in mouse,rat, non-human primates, canines, goat, rabbit, porcine, feline, andhorses. In other words, a carbohydrate transporter molecule or acarbohydrate metabolic enzyme molecule can comprise a nucleic acidsequence homologous to the human nucleic acid that encodes a human GLUT2and SGLT1 protein, or human sucrase-isomaltase (SI), humanmaltase-glucoamylase, and human lactase protein, respectively, whereinthe nucleic acid is found in a different species and wherein thathomolog encodes a protein with a glucose transporter function similar toa carbohydrate transporter molecule or an enzymatic function similar toa carbohydrate metabolic enzyme molecule.

A carbohydrate transporter molecule of this invention also encompassesvariants of the human nucleic acid encoding the GLUT2 or SGLT1 proteinsthat exhibit carbohydrate transporter activity, or variants of the humanGLUT2 or SGLT1 proteins that exhibit carbohydrate transporter activity.A carbohydrate transporter molecule of this invention also includes afragment of the human GLUT2 or SGLT1 nucleic acid which encodes apolypeptide that exhibits carbohydrate transporter activity. Acarbohydrate transporter molecule of this invention encompasses afragment of the human GLUT2 or SGLT1 protein that exhibits carbohydratetransporter activity.

A carbohydrate metabolic enzyme molecule of this invention alsoencompasses variants of the human nucleic acid encoding thesucrase-isomaltase (SI), human maltase-glucoamylase, and human lactaseproteins that exhibit carbohydrate metabolic enzyme activity, orvariants of the human sucrase-isomaltase (SI), humanmaltase-glucoamylase, and human lactase proteins that exhibitcarbohydrate metabolic enzyme activity. A carbohydrate metabolic enzymemolecule of this invention also includes a fragment of the humansucrase-isomaltase (SI), human maltase-glucoamylase, and human lactasenucleic acid which encodes a polypeptide that exhibits carbohydratemetabolic enzyme activity. A carbohydrate metabolic enzyme molecule ofthis invention encompasses a fragment of the human sucrase-isomaltase(SI), human maltase-glucoamylase, and human lactase protein thatexhibits carbohydrate metabolic enzyme activity.

The variants can comprise, for instance, naturally-occurring variantsdue to allelic variations between individuals (e.g., polymorphisms),mutated alleles related to autism, or alternative splicing forms. In oneembodiment, a carbohydrate transporter molecule is a nucleic acidvariant of the nucleic acid having the sequence shown in SEQ ID NO: 2,wherein the variant has a nucleotide sequence identity to SEQ ID NO: 2of at least about 65%, at least about 75%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% with SEQ ID NO: 2.In another embodiment, a carbohydrate transporter molecule is a nucleicacid variant of the nucleic acid having the sequence shown in SEQ ID NO:4, wherein the variant has a nucleotide sequence identity to SEQ ID NO:4 of at least about 65%, at least about 75%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% with SEQ IDNO: 4. In one embodiment, a carbohydrate metabolic enzyme molecule is anucleic acid variant of the nucleic acid having the sequence shown inSEQ ID NO: 6, wherein the variant has a nucleotide sequence identity toSEQ ID NO: 6 of at least about 65%, at least about 75%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99% withSEQ ID NO: 6. In another embodiment, a carbohydrate metabolic enzymemolecule is a nucleic acid variant of the nucleic acid having thesequence shown in SEQ ID NO: 8, wherein the variant has a nucleotidesequence identity to SEQ ID NO: 8 of at least about 65%, at least about75%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% with SEQ ID NO: 8. In a further embodiment, acarbohydrate metabolic enzyme molecule is a nucleic acid variant of thenucleic acid having the sequence shown in SEQ ID NO: 10, wherein thevariant has a nucleotide sequence identity to SEQ ID NO: 10 of at leastabout 65%, at least about 75%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% with SEQ ID NO: 10.

In one embodiment, a carbohydrate transporter molecule encompasses anyportion of at least about 8 consecutive nucleotides of SEQ ID NO: 2 or4. In one embodiment, the fragment can comprise at least about 15nucleotides, at least about 20 nucleotides, or at least about 30nucleotides of SEQ ID NO: 2 or 4. Fragments include all possiblenucleotide lengths between about 8 and 100 nucleotides, for example,lengths between about 15 and 100, or between about 20 and 100. In oneembodiment, a carbohydrate metabolic enzyme molecule encompasses anyportion of at least about 8 consecutive nucleotides of SEQ ID NO: 6, 8,or 10. In one embodiment, the fragment can comprise at least about 15nucleotides, at least about 20 nucleotides, or at least about 30nucleotides of SEQ ID NO: 6, 8, or 10. Fragments include all possiblenucleotide lengths between about 8 and 100 nucleotides, for example,lengths between about 15 and 100, or between about 20 and 100.

The invention further provides for nucleic acids that are complementaryto a nucleic acid encoding GLUT2, SGLT1, sucrase-isomaltase (SI), humanmaltase-glucoamylase, or human lactase proteins. Such complementarynucleic acids can comprise nucleic acid sequences, which hybridize to anucleic acid sequence encoding a GLUT2, SGLT1, sucrase-isomaltase (SI),maltase-glucoamylase, or lactase protein under stringent hybridizationconditions. Non-limiting examples of stringent hybridization conditionsinclude temperatures above 30° C., above 35° C., in excess of 42° C.,and/or salinity of less than about 500 mM, or less than 200 mM.Hybridization conditions can be adjusted by the skilled artisan viamodifying the temperature, salinity and/or the concentration of otherreagents such as SDS or SSC.

In one embodiment, a carbohydrate transporter molecule comprises aprotein or polypeptide encoded by a carbohydrate transporter nucleicacid sequence, such as the sequence shown in SEQ ID NO: 2 or 4. Inanother embodiment, the polypeptide can be modified, such as byglycosylations and/or acetylations and/or chemical reaction or coupling,and can contain one or several non-natural or synthetic amino acids. Anexample of a carbohydrate transporter molecule is the polypeptide havingthe amino acid sequence shown in SEQ ID NO: 1 or 3. In one embodiment, acarbohydrate metabolic enzyme molecule comprises a protein orpolypeptide encoded by a carbohydrate metabolic enzyme nucleic acidsequence, such as the sequence shown in SEQ ID NO: 6, 8, or 10. Inanother embodiment, the polypeptide can be modified, such as byglycosylations and/or acetylations and/or chemical reaction or coupling,and can contain one or several non-natural or synthetic amino acids. Anexample of a carbohydrate transporter molecule is the polypeptide havingthe amino acid sequence shown in SEQ ID NO: 5, 7, or 9.

In another embodiment, a carbohydrate transporter molecule can be afragment of a carbohydrate transporter protein, such as GLUT2 or SGLT1.For example, the carbohydrate transporter molecule can encompass anyportion of at least about 8 consecutive amino acids of SEQ ID NO: 1 or3. The fragment can comprise at least about 10 amino acids, a leastabout 20 amino acids, at least about 30 amino acids, at least about 40amino acids, a least about 50 amino acids, at least about 60 aminoacids, or at least about 75 amino acids of SEQ ID NO: 1 or 3. In anotherembodiment, a carbohydrate metabolic enzyme molecule can be a fragmentof a carbohydrate metabolic enzyme protein, such as sucrase-isomaltase(SI), maltase-glucoamylase, or lactase. For example, the carbohydratemetabolic enzyme molecule can encompass any portion of at least about 8consecutive amino acids of SEQ ID NO: 5, 7, or 9. The fragment cancomprise at least about 10 amino acids, a least about 20 amino acids, atleast about 30 amino acids, at least about 40 amino acids, a least about50 amino acids, at least about 60 amino acids, or at least about 75amino acids of SEQ ID NO: 5, 7, or 9. Fragments include all possibleamino acid lengths between about 8 and 100 about amino acids, forexample, lengths between about 10 and 100 amino acids, between about 15and 100 amino acids, between about 20 and 100 amino acids, between about35 and 100 amino acids, between about 40 and 100 amino acids, betweenabout 50 and 100 amino acids, between about 70 and 100 amino acids,between about 75 and ,100 amino acids, or between about 80 and 100 aminoacids.

In certain embodiments, the carbohydrate transporter molecule of theinvention includes variants of the human GLUT2 or SGLT1 protein (havingthe amino acid sequence shown in SEQ ID NO: 1 and 3, respectively). Suchvariants can include those having at least from about 46% to about 50%identity to SEQ ID NO: 1 or 3, or having at least from about 50.1% toabout 55% identity to SEQ ID NO: 1 or 3, or having at least from about55.1% to about 60% identity to SEQ ID NO: 1 or 3, or having from atleast about 60.1% to about 65% identity to SEQ ID NO: 1 or 3, or havingfrom about 65.1% to about 70% identity to SEQ ID NO: 1 or 3, or havingat least from about 70.1% to about 75% identity to SEQ ID NO: 1 or 3, orhaving at least from about 75.1% to about 80% identity to SEQ ID NO: 1or 3, or having at least from about 80.1% to about 85% identity to SEQID NO: 1 or 3, or having at least from about 85.1% to about 90% identityto SEQ ID NO: 1 or 3, or having at least from about 90.1% to about 95%identity to SEQ ID NO: 1 or 3, or having at least from about 95.1% toabout 97% identity to SEQ ID NO: 1 or 3, or having at least from about97.1% to about 99% identity to SEQ ID NO: 1 or 3.

In certain embodiments, the carbohydrate metabolic enzyme molecule ofthe invention includes variants of the human sucrase-isomaltase (SI),maltase-glucoamylase, or lactase protein (having the amino acid sequenceshown in SEQ ID NO: 5, 7, and 9, respectively). Such variants caninclude those having at least from about 46% to about 50% identity toSEQ ID NO: 5, 7, or 9, or having at least from about 50.1% to about 55%identity to SEQ ID NO: 5, 7, or 9, or having at least from about 55.1%to about 60% identity to SEQ ID NO: 5, 7, or 9, or having from at leastabout 60.1% to about 65% identity to SEQ ID NO: 5, 7, or 9, or havingfrom about 65.1% to about 70% identity to SEQ ID NO: 5, 7, or 9, orhaving at least from about 70.1% to about 75% identity to SEQ ID NO: 5,7, or 9, or having at least from about 75.1% to about 80% identity toSEQ ID NO: 5, 7, or 9, or having at least from about 80.1% to about 85%identity to SEQ ID NO: 5, 7, or 9, or having at least from about 85.1%to about 90% identity to SEQ ID NO: 5, 7, or 9, or having at least fromabout 90.1% to about 95% identity to SEQ ID NO: 5, 7, or 9, or having atleast from about 95.1% to about 97% identity to SEQ ID NO: 5, 7, or 9,or having at least from about 97.1% to about 99% identity to SEQ ID NO:5, 7, or 9.

In another embodiment, the carbohydrate transporter molecule of theinvention encompasses a peptidomimetic which exhibits carbohydratetransporter activity. In another embodiment, the carbohydratetransporter molecule of the invention encompasses a peptidomimetic whichexhibits carbohydrate transporter activity. In another embodiment, thecarbohydrate metabolic enzyme molecule of the invention encompasses apeptidomimetic which exhibits carbohydrate metabolic enzyme activity. Inanother embodiment, the carbohydrate metabolic enzyme molecule of theinvention encompasses a peptidomimetic which exhibits carbohydratemetabolic enzyme activity. A peptidomimetic is a small protein-likechain designed to mimic a peptide that can arise from modification of anexisting peptide in order to protect that molecule from enzymedegradation and increase its stability, and/or alter the molecule'sproperties (for example modifications that change the molecule'sstability or biological activity). These modifications involve changesto the peptide that can not occur naturally (such as altered backbonesand the incorporation of non-natural amino acids). Drug-like compoundscan be able to be developed from existing peptides. A peptidomimetic canbe a peptide, partial peptide or non-peptide molecule that mimics thetertiary binding structure or activity of a selected native peptide orprotein functional domain (e.g., binding motif or active site). Thesepeptide mimetics include recombinantly or chemically modified peptides.

In one embodiment, a carbohydrate transporter molecule comprising SEQ IDNO: 1, SEQ ID NO: 3, variants of each, or fragments thereof, can bemodified to produce peptide mimetics by replacement of one or morenaturally occurring side chains of the 20 genetically encoded aminoacids (or D amino acids) with other side chains. In one embodiment, acarbohydrate metabolic enzyme molecule comprising SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, variants of, or fragments thereof, can be modifiedto produce peptide mimetics by replacement of one or more naturallyoccurring side chains of the 20 genetically encoded amino acids (or Damino acids) with other side chains. This can occur, for instance, withgroups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-memberedalkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,hydroxy, carboxy and the lower ester derivatives thereof, and with 4,5-, 6-, to 7-membered heterocyclics. For example, proline analogs can bemade in which the ring size of the proline residue is changed from 5members to 4, 6, or 7 members. Cyclic groups can be saturated orunsaturated, and if unsaturated, can be aromatic or non-aromatic.Heterocyclic groups can contain one or more nitrogen, oxygen, and/orsulphur heteroatoms. Examples of such groups include the furazanyl,ifuryl, imidazolidinyl imidazolyl, imidazolinyl, isothiazolyl,isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino),and triazolyl. These heterocyclic groups can be substituted orunsubstituted. Where a group is substituted, the substituent can bealkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.Peptidomimetics can also have amino acid residues that have, beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties. For example, peptidomimeticscan be designed and directed to amino acid sequences encoded by acarbohydrate transporter molecule comprising SEQ ID NO: 1 or 3. Forexample, peptidomimetics can be designed and directed to amino acidsequences encoded by a carbohydrate metabolic enzyme molecule comprisingSEQ ID NO: 5, 7, or 9.

A variety of techniques are available for constructing peptide mimeticswith the same or similar desired biological activity as thecorresponding native but with more favorable activity than the peptidewith respect to solubility, stability, and/or susceptibility tohydrolysis or proteolysis (see, e.g., Morgan & Gainor, Ann. Rep. Med.Chem. 24,243-252, 1989). Certain peptidomimetic compounds are based uponthe amino acid sequence of the peptides of the invention. Peptidomimeticcompounds can be synthetic compounds having a three-dimensionalstructure (i.e. a peptide motif) based upon the three-dimensionalstructure of a selected peptide. The peptide motif provides thepeptidomimetic compound with the desired biological activity, whereinthe binding activity of the mimetic compound is not substantiallyreduced, and is often the same as or greater than the activity of thenative peptide on which the mimetic is modeled. Peptidomimetic compoundscan have additional characteristics that enhance their therapeuticapplication, such as increased cell permeability, greater affinityand/or avidity and prolonged biological half-life. Peptidomimetic designstrategies are readily available in the art (see, e.g., Ripka & Rich,Curr. Op. Chem. Biol. 2, 441452, 1998; Hrubyet al., Curr. Op. Chem.Biol. 1, 114119, 1997; Hruby & Balse, Curr. Med. Chem. 9,945-970,-2000).

Diagnosis

The invention provides diagnosis methods based on monitoring a geneencoding a carbohydrate metabolic enzyme molecule (such as sucraseisomaltase, maltase glucoamylase, or lactase) or a carbohydratetransporter molecule (such as GLUT2 or SGLT1). As used herein, the term“diagnosis” includes the detection, typing, monitoring, dosing,comparison, at various stages, including early, pre-symptomatic stages,and late stages, in adults, children, and unborn human children.Diagnosis can include the assessment of a predisposition or risk ofdevelopment, the prognosis, or the characterization of a subject todefine most appropriate treatment (pharmacogenetics).

The invention provides diagnostic methods to determine whether anindividual is at risk of developing autism or an autism spectrumdisorder (ASD), or suffers from autism or an ASD, wherein the diseasereflects an alteration in the expression of a gene encoding acarbohydrate metabolic enzyme molecule (such as sucrase isomaltase,maltase glucoamylase, or lactase) or a carbohydrate transporter molecule(such as GLUT2 or SGLT1). Subjects diagnosed with autism, as well asASD, can display some core symptoms in the areas of a) socialinteractions and relationships, b) verbal and non-verbal communication,and c) physical activity, play, physical behavior. For example, symptomsrelated to social interactions and relationships can include but are notlimited to the inability to establish friendships with children the sameage, lack of empathy, and the inability to develop nonverbalcommunicative skills (for example, eye-to-eye gazing, facialexpressions, and body posture). For example, symptoms related to verbaland nonverbal communication comprises delay in learning to talk,inability to learn to talk, failure to initiate or maintain aconversation, failure to interpret or understand implied meaning ofwords, and repetitive use of language. For example, symptoms related tophysical activity, play, physical behavior include, but are not limitedto unusual focus on pieces or parts of an object, such as a toy, apreoccupation with certain topics, a need for routines and rituals, andstereotyped behaviors (for example, body rocking and hand flapping).

In one embodiment, a method of detecting the presence of or apredisposition to autism or an autism spectrum disorder in a subject isprovided. The subject can be a human or a child thereof. The subject canalso be a human embryo, a human fetus, or an unborn human child. Themethod can comprise detecting in a sample from the subject the presenceof an alteration in the expression of a gene of a carbohydrate metabolicenzyme molecule (such as sucrase isomaltase, maltase glucoamylase, orlactase) or a carbohydrate transporter molecule (such as GLUT2 orSGLT1). In one embodiment, the detecting comprises detecting whetherthere is an alteration in the gene locus encoding a carbohydratemetabolic enzyme molecule (such as sucrase isomaltase, maltaseglucoamylase, or lactase) or a carbohydrate transporter molecule (suchas GLUT2 or SGLT1). In a further embodiment, the detecting comprisesdetecting whether expression of a carbohydrate metabolic enzyme molecule(such as sucrase isomaltase, maltase glucoamylase, or lactase) or acarbohydrate transporter molecule (such as GLUT2 or SGLT1) is reduced.In some embodiments, the detecting comprises detecting in the samplewhether there is a reduction in an mRNA encoding a carbohydratemetabolic enzyme molecule or a carbohydrate transporter molecule, or areduction in either the carbohydrate metabolic enzyme protein or acarbohydrate transporter protein, or a combination thereof. The presenceof such an alteration is indicative of the presence or predisposition toautism or an autism spectrum disorder. The presence of an alteration ina gene encoding a carbohydrate metabolic enzyme molecule or acarbohydrate transporter molecule in the sample is detected through thegenotyping of a sample, for example via gene sequencing, selectivehybridization, amplification, gene expression analysis, or a combinationthereof. In one embodiment, the sample can comprise blood, serum,sputum, lacrimal secretions, semen, vaginal secretions, fetal tissue,skin tissue, ileum tissue, cecum tissue, muscle tissue, amniotic fluid,or a combination thereof.

The invention also provides a method for treating or preventing autismor an autism spectrum disorder in a subject. In one embodiment, themethod comprises (1) detecting the presence of an alteration in acarbohydrate transporter gene or a carbohydrate metabolic enzyme in asample from the subject, where the presence of the alteration isindicative of autism or an ASD, or the predisposition to autism or ASD,and, (2) administering to the subject in need a therapeutic treatmentagainst autism or an autism spectrum disorder. In one embodiment, thecarbohydrate transporter gene can be a GLUT2 gene or a SGLT1 gene. Inanother embodiment, the carbohydrate metabolic enzyme gene can be asucrase isomaltase gene, a maltase glucoamylase gene, or a lactase gene.The therapeutic treatment can be a drug administration (for example, apharmaceutical composition comprising a functional carbohydratetransporter molecule or a functional carbohydrate metabolic enzymemolecule). In one embodiment, the molecule comprises a carbohydratetransporter polypeptide comprising at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 93%, atleast about 95%, at least about 97%, at least about 98%, at least about99%, or 100% of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3,and exhibits the function of restoring functional carbohydratetransporter expression in deficient individuals, thus restoring thecapacity for carbohydrate transport. In another embodiment, the moleculecomprises a carbohydrate metabolic enzyme polypeptide comprising atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 93%, at least about 95%, at least about 97%, atleast about 98%, at least about 99%, or 100% of the amino acid sequenceof SEQ ID NO: 5, 7, or 9, and exhibits the function of restoringfunctional carbohydrate metabolic enzyme expression in deficientindividuals, thus restoring the capacity for carbohydrate metabolism.

In some embodiments, the molecule comprises a nucleic acid encoding acarbohydrate transporter polypeptide comprising at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about93%, at least about 95%, at least about 97%, at least about 98%, atleast about 99%, or 100% of the nucleic acid sequence of SEQ ID NO: 2 or4 and encodes a polypeptide with the function of restoring functionalcarbohydrate transporter expression in deficient individuals, thusrestoring the capacity for carbohydrate transport. In furtherembodiments, the molecule comprises a nucleic acid encoding acarbohydrate metabolic enzyme polypeptide comprising at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 93%, at least about 95%, at least about 97%, at least about 98%,at least about 99%, or 100% of the nucleic acid sequence of SEQ ID NO:6, 8, or 10, and encodes a polypeptide with the function of restoringfunctional carbohydrate metabolic enzyme expression in deficientindividuals, thus restoring the capacity for carbohydrate metabolism.

The alteration can be determined at the DNA, RNA or polypeptide level ofthe carbohydrate transporter or carbohydrate metabolic enzyme. Thedetection can also be determined by performing an oligonucleotideligation assay, a confirmation based assay, a hybridization assay, asequencing assay, an allele-specific amplification assay, amicrosequencing assay, a melting curve analysis, a denaturing highperformance liquid chromatography (DHPLC) assay (for example, see Joneset al, (2000) Hum Genet., 106(6):663-8), or a combination thereof. Insome embodiments, the detection is performed by sequencing all or partof a carbohydrate transporter or carbohydrate metabolic enzyme gene orby selective hybridization or amplification of all or part of acarbohydrate transporter or carbohydrate metabolic enzyme gene. Acarbohydrate transporter or carbohydrate metabolic enzyme gene specificamplification can be carried out before the alteration identificationstep.

An alteration in a carbohydrate transporter gene locus (e.g., whereGLUT2 or SGLT1 are located) or a carbohydrate metabolic enzyme genelocus (e.g., where SI, MGAM, or LCT are located) can be any form ofmutation(s), deletion(s), rearrangement(s) and/or insertions in thecoding and/or non-coding region of the locus, alone or in variouscombination(s). Mutations can include point mutations. Insertions canencompass the addition of one or several residues in a coding ornon-coding portion of the gene locus. Insertions can comprise anaddition of between 1 and 50 base pairs in the gene locus. Deletions canencompass any region of one, two or more residues in a coding ornon-coding portion of the gene locus, such as from two residues up tothe entire gene or locus. Deletions can affect smaller regions, such asdomains (introns) or repeated sequences or fragments of less than about50 consecutive base pairs, although larger deletions can occur as well.Rearrangement includes inversion of sequences. The carbohydratetransporter gene locus alteration or carbohydrate metabolic enzyme genelocus alteration can result in amino acid substitutions, RNA splicing orprocessing, product instability, the creation of stop codons,frame-shift mutations, and/or truncated polypeptide production. Thealteration can result in the production of a carbohydrate transporterpolypeptide or a carbohydrate metabolic enzyme with altered function,stability, targeting or structure. The alteration can also cause areduction in protein expression. In one embodiment, the alteration in acarbohydrate transporter gene locus can comprise a point mutation, adeletion, or an insertion in the carbohydrate transporter gene orcorresponding expression product. In another embodiment, the alterationin a carbohydrate metabolic enzyme gene locus can comprise a pointmutation, a deletion, or an insertion in the carbohydrate metabolicenzyme gene or corresponding expression product. In one embodiment, thealteration can be a deletion or partial deletion of a carbohydratetransporter gene or a carbohydrate metabolic enzyme gene. The alterationcan be determined at the level of the DNA, RNA, or polypeptide of acarbohydrate transporter or a carbohydrate metabolic enzyme.

In another embodiment, the method can comprise detecting the presence ofan altered RNA expression of a carbohydrate transporter or acarbohydrate metabolic enzyme. Altered RNA expression includes thepresence of an altered RNA sequence, the presence of an altered RNAsplicing or processing, or the presence of an altered quantity of RNA.These can be detected by various techniques known in the art, includingby sequencing all or part of the RNA of a carbohydrate transporter or acarbohydrate metabolic enzyme, or by selective hybridization orselective amplification of all or part of the RNA. In a furtherembodiment, the method can comprise detecting the presence of an alteredpolypeptide expression of a carbohydrate transporter or a carbohydratemetabolic enzyme. Altered polypeptide expression includes the presenceof an altered polypeptide sequence, the presence of an altered quantityof carbohydrate transporter polypeptide or carbohydrate metabolic enzymepolypeptide, or the presence of an altered tissue distribution. Thesecan be detected by various techniques known in the art, including bysequencing and/or binding to specific ligands (such as antibodies).

Various techniques known in the art can be used to detect or quantifyaltered gene expression, RNA expression, or sequence, which include, butare not limited to, hybridization, sequencing, amplification, and/orbinding to specific ligands (such as antibodies). Other suitable methodsinclude allele-specific oligonucleotide (ASO), oligonucleotide ligation,allele-specific amplification, Southern blot (for DNAs), Northern blot(for RNAs), single-stranded conformation analysis (SSCA), PFGE,fluorescent in situ hybridization (FISH), gel migration, clampeddenaturing gel electrophoresis, denaturing HLPC, melting curve analysis,heteroduplex analysis, RNase protection, chemical or enzymatic mismatchcleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays(IEMA). Some of these approaches (such as SSCA and CGGE) are based on achange in electrophoretic mobility of the nucleic acids, as a result ofthe presence of an altered sequence. According to these techniques, thealtered sequence is visualized by a shift in mobility on gels. Thefragments can then be sequenced to confirm the alteration. Some otherapproaches are based on specific hybridization between nucleic acidsfrom the subject and a probe specific for wild type or altered gene orRNA. The probe can be in suspension or immobilized on a substrate. Theprobe can be labeled to facilitate detection of hybrids. Some of theseapproaches are suited for assessing a polypeptide sequence or expressionlevel, such as Northern blot, ELISA and RIA. These latter require theuse of a ligand specific for the polypeptide, for example, the use of aspecific antibody.

Sequencing. Sequencing can be carried out using techniques well known inthe art, using automatic sequencers. The sequencing can be performed onthe complete gene or on specific domains thereof, such as those known orsuspected to carry deleterious mutations or other alterations.

Amplification. Amplification is based on the formation of specifichybrids between complementary nucleic acid sequences that serve toinitiate nucleic acid reproduction. Amplification can be performedaccording to various techniques known in the art, such as by polymerasechain reaction (PCR), ligase chain reaction (LCR), strand displacementamplification (SDA) and nucleic acid sequence based amplification(NASBA). These techniques can be performed using commercially availablereagents and protocols. Useful techniques in the art encompass real-timePCR, allele-specific PCR, or PCR-SSCP. Amplification usually requiresthe use of specific nucleic acid primers, to initiate the reaction. Forexample, nucleic acid primers useful for amplifying sequences from thegene or locus of a carbohydrate transporter (such as GLUT2 or SGLT1) ora carbohydrate metabolic enzyme (such as SI, MGAM, or LCT) are able tospecifically hybridize with a portion of the gene locus that flanks atarget region of the locus, wherein the target region is altered incertain subjects having autism or an autism spectrum disorder. In oneembodiment, amplification comprises using forward and reverse RT-PCRprimers comprising nucleotide sequences of SEQ ID NOS: 26, 27, 29, 30,32, 33, 35, 36, 38, or 39.

The invention provides for a nucleic acid primer, wherein the primer canbe complementary to and hybridize specifically to a portion of a codingsequence (e.g., gene or RNA) of a carbohydrate transporter (such asGLUT2 or SGLT1) or a carbohydrate metabolic enzyme (such as SI, MGAM, orLCT) that is altered in certain subjects having autism or an autismspectrum disorder. Primers of the invention can thus be specific foraltered sequences in a gene or RNA of a carbohydrate transporter or acarbohydrate metabolic enzyme. By using such primers, the detection ofan amplification product indicates the presence of an alteration in thegene or the absence of such gene. Examples of primers of this inventioncan be single-stranded nucleic acid molecules of about 5 to 60nucleotides in length, or about 8 to about 25 nucleotides in length. Thesequence can be derived directly from the sequence of the carbohydratetransporter or the carbohydrate metabolic enzyme gene (e.g., GLUT2 orSGLT1, and SI, MGAM, or LCT, respectively). Perfect complementarity isuseful, to ensure high specificity. However, certain mismatch can betolerated. In one embodiment, the primer can be an isolated nucleic acidcomprising a nucleotide sequence of SEQ ID NOS: 26, 27, 29, 30, 32, 33,35, 36, 38, or 39. For example, a nucleic acid primer or a pair ofnucleic acid primers as described above can be used in a method fordetecting the presence of or a predisposition to autism or an autismspectrum disorder in a subject.

Amplification methods include, e.g., polymerase chain reaction, PCR (PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560,1989; Landegren, Science 241:1077, 1988; Barringer, Gene 89:117, 1990);transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad. Sci. USA86:1173, 1989); and, self-sustained sequence replication (see, e.g.,Guatelli, Proc. Natl. Acad. Sci. USA 87:1874, 1990); Q Beta replicaseamplification (see, e.g., Smith, J. Clin. Microbiol. 35:1477-1491,1997), automated Q-beta replicase amplification assay (see, e.g., Burg,Mol. Cell. Probes 10:257-271, 1996) and other RNA polymerase mediatedtechniques (e.g., NASBA, Cangene, Mississauga, Ontario); see alsoBerger, Methods Enzymol. 152:307-316, 1987; Sambrook; Ausubel; U.S. Pat.Nos. 4,683,195 and 4,683,202; Sooknanan, Biotechnology 13:563-564, 1995.All the references stated above are incorporated by reference in theirentireties.

Selective Hybridization. Hybridization detection methods are based onthe formation of specific hybrids between complementary nucleic acidsequences that serve to detect nucleic acid sequence alteration(s). Adetection technique involves the use of a nucleic acid probe specificfor wild type or altered gene or RNA, followed by the detection of thepresence of a hybrid. The probe can be in suspension or immobilized on asubstrate or support (for example, as in nucleic acid array or chipstechnologies). The probe can be labeled to facilitate detection ofhybrids. In one embodiment, the probe according to the invention cancomprise a nucleic acid having SEQ ID NOS: 28, 31, 34, 37, or 40. Forexample, a sample from the subject can be contacted with a nucleic acidprobe specific for a wild type carbohydrate transporter or carbohydratemetabolic enzyme gene or an altered carbohydrate transporter orcarbohydrate metabolic enzyme gene, and the formation of a hybrid can besubsequently assessed. In one embodiment, the method comprisescontacting simultaneously the sample with a set of probes that arespecific, respectively, for the wild type carbohydrate transporter orcarbohydrate metabolic enzyme gene and for various altered formsthereof. Thus, it is possible to detect directly the presence of variousforms of alterations in the carbohydrate transporter gene (e.g., GLUT2or SGLT1) or carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or LCT)in the sample. Also, various samples from various subjects can betreated in parallel.

According to the invention, a probe can be a polynucleotide sequencewhich is complementary to and specifically hybridizes with a, or atarget portion of a, carbohydrate transporter or carbohydrate metabolicenzyme gene or RNA, and that is suitable for detecting polynucleotidepolymorphisms associated with alleles of such, which predispose to orare associated with autism or an autism spectrum disorder. Useful probesare those that are complementary to the carbohydrate transporter orcarbohydrate metabolic enzyme gene, RNA, or target portion thereof.Probes can comprise single-stranded nucleic acids of between 8 to 1000nucleotides in length, for instance between 10 and 800, between. 15 and700, or between 20 and 500. Longer probes can be used as well. A usefulprobe of the invention is a single stranded nucleic acid molecule ofbetween 8 to 500 nucleotides in length, which can specifically hybridizeto a region of a gene or RNA that carries an alteration.

The sequence of the probes can be derived from the sequences of thecarbohydrate transporter or carbohydrate metabolic enzyme genes providedherein. Nucleotide substitutions can be performed, as well as chemicalmodifications of the probe. Such chemical modifications can beaccomplished to increase the stability of hybrids (e.g., intercalatinggroups) or to label the probe. Some examples of labels include, withoutlimitation, radioactivity, fluorescence, luminescence, and enzymaticlabeling.

A guide to the hybridization of nucleic acids is found in e.g.,Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2^(ND) ED.),Vols. 1-3, Cold Spring Harbor Laboratory, 1989; CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York, 1997;LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:HYBRIDIZATION WITH NUCLEIC ACID PROBES, PART I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y., 1993.

Specific Ligand Binding. As indicated herein, alteration in acarbohydrate transporter or carbohydrate metabolic enzyme gene locus orin carbohydrate transporter or carbohydrate metabolic enzyme expressioncan also be detected by screening for alteration(s) in correspondingpolypeptide sequence or expression levels. Different types of ligandscan be used, such as specific antibodies. In one embodiment, the sampleis contacted with an antibody specific for a carbohydrate transporter orcarbohydrate metabolic enzyme polypeptide and the formation of an immunecomplex is subsequently determined. Various methods for detecting animmune complex can be used, such as ELISA, radioimmunoassays (RIA) andimmuno-enzymatic assays (IEMA).

For example, an antibody can be a polyclonal antibody, a monoclonalantibody, as well as fragments or derivatives thereof havingsubstantially the same antigen specificity. Fragments include Fab,Fab′2, or CDR regions. Derivatives include single-chain antibodies,humanized antibodies, or poly-functional antibodies. An antibodyspecific for a carbohydrate transporter or a carbohydrate metabolicenzyme polypeptide can be an antibody that selectively binds acarbohydrate transporter or carbohydrate metabolic enzyme polypeptide,respectively, namely, an antibody raised against a carbohydratetransporter or carbohydrate metabolic enzyme polypeptide or anepitope-containing fragment thereof. Although non-specific bindingtowards other antigens can occur, binding to the target polypeptideoccurs with a higher affinity and can be reliably discriminated fromnon-specific binding. In one embodiment, the method comprises contactinga sample from the subject with an antibody specific for a wild type oran altered form of a carbohydrate transporter or carbohydrate metabolicenzyme polypeptide, and determining the presence of an immune complex.Optionally, the sample can be contacted to a support coated withantibody specific for the wild type or altered form of a carbohydratetransporter or carbohydrate metabolic enzyme polypeptide. In oneembodiment, the sample can be contacted simultaneously, or in parallel,or sequentially, with various antibodies specific for different forms ofa carbohydrate transporter or carbohydrate metabolic enzyme polypeptide,such as a wild type and various altered forms thereof.

The invention also provides for a diagnostic kit comprising products andreagents for detecting in a sample from a subject the presence of analteration in a carbohydrate transporter gene (e.g., GLUT2 or SGLT1) ora carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or LCT), or acarbohydrate transporter polypeptide or carbohydrate metabolic enzymepolypeptide; alteration in the expression of a carbohydrate transportergene (e.g., GLUT2 or SGLT1) or carbohydrate metabolic enzyme gene (e.g.,SI, MGAM, or LCT), or a carbohydrate transporter or carbohydratemetabolic enzyme polypeptide; and/or an alteration in carbohydratetransporter or carbohydrate metabolic enzyme activity. The kit can beuseful for determining whether a sample from a subject exhibits reducedcarbohydrate transporter or carbohydrate metabolic enzyme expression orexhibits a gene deletion of a carbohydrate transporter (e.g., GLUT2 orSGLT1) or carbohydrate metabolic enzyme (e.g., SI, MGAM, or LCT). Forexample, the diagnostic kit according to the present invention comprisesany primer, any pair of primers, any nucleic acid probe and/or anyligand, (for example, an antibody directed to a carbohydrate transporteror carbohydrate metabolic enzyme). The diagnostic kit according to thepresent invention can further comprise reagents and/or protocols forperforming a hybridization, amplification or antigen-antibody immunereaction. In one embodiment, the kit can comprise nucleic acid primersthat specifically hybridize to and can prime a polymerase reaction froma carbohydrate transporter (e.g., GLUT2 or SGLT1) or carbohydratemetabolic enzyme (e.g., SI, MGAM, or LCT). In another embodiment, theprimer can comprise a nucleotide sequence of SEQ ID NOS: 26, 27, 29, 30,32, 33, 35, 36, 38, or 39.

The diagnosis methods can be performed in vitro, ex vivo, or in vivo.These methods utilize a sample from the subject in order to assess thestatus of a carbohydrate transporter gene locus or a carbohydratemetabolic enzyme gene locus. The sample can be any biological samplederived from a subject, which contains nucleic acids or polypeptides.Examples of such samples include, but are not limited to, fluids,tissues, cell samples, organs, or tissue biopsies. Non-limiting examplesof samples include blood, plasma, saliva, urine, or seminal fluid.Pre-natal diagnosis can also be performed by testing fetal cells orplacental cells, for instance. Screening of parental samples can also beused to determine risk/likelihood of offspring possessing the germlinemutation. The sample can be collected according to conventionaltechniques and used directly for diagnosis or stored. The sample can betreated prior to performing the method, in order to render or improveavailability of nucleic acids or polypeptides for testing. Treatmentsinclude, for instance, lysis (e.g., mechanical, physical, or chemical),centrifugation. Also, the nucleic acids and/or polypeptides can bepre-purified or enriched by conventional techniques, and/or reduced incomplexity. Nucleic acids and polypeptides can also be treated withenzymes or other chemical or physical treatments to produce fragmentsthereof. In one embodiment, the sample is contacted with reagents, suchas probes, primers, or ligands, in order to assess the presence of analtered carbohydrate transporter gene locus or carbohydrate metabolicenzyme gene locus. Contacting can be performed in any suitable device,such as a plate, tube, well, or glass. In specific embodiments, thecontacting is performed on a substrate coated with the reagent, such asa nucleic acid array or a specific ligand array. The substrate can be asolid or semi-solid substrate such as any support comprising glass,plastic, nylon, paper, metal, or polymers. The substrate can be ofvarious forms and sizes, such as a slide, a membrane, a bead, a column,or a gel. The contacting can be made under any condition suitable for acomplex to be formed between the reagent and the nucleic acids orpolypeptides of the sample.

Identifying an altered polypeptide, RNA or DNA of a carbohydratetransporter (e.g., GLUT2 or SGLT1) or a carbohydrate metabolic enzyme(e.g., SI, MGAM, or LCT) in the sample is indicative of the presence ofan altered carbohydrate transporter or carbohydrate metabolic enzymegene in the subject, which can be correlated to the presence,predisposition or stage of progression of autism or an autism spectrumdisorder. For example, an individual having a germ line mutation in acarbohydrate transporter gene (e.g., GLUT 2 or SGLT1) or a carbohydratemetabolic enzyme gene (e.g., SI, MGAM, or LCT) has an increased risk ofdeveloping autism or an autism spectrum disorder. The determination ofthe presence of an altered gene locus in a subject also allows thedesign of appropriate therapeutic intervention, which is more effectiveand customized. Also, this determination at the pre-symptomatic levelallows a preventive regimen to be applied.

GI Bacterial Colonization in ASD Subjects

An aspect of the invention provides for a new PCR strategy for theidentification, quantitation, and taxonomic classification of Sutterellabacterial colonization from biological samples. As shown in Example 2herein, intestinal biopsies of children with autism accompanied bygastrointestinal (GI) complaints showed highly significant elevation ofintestinal levels of Sutterella bacteria. These findings can provideinsights into pathogenesis of autism associated with GI disorder,enabling new strategies for therapeutic intervention.

Bacterial members of the genus Sutterella, a class ofBeta-proteobacteria in the order Burkholderiales and the familyAlcaligenaceae have been associated with human infections below thediaphragm (A1). Furthermore, Sutterella sp. sequences have beenidentified in intestinal biopsies and fecal samples from individualswith Crohn's disease and ulcerative colitis (A2, A3). Sutterella sp.have also been found in canine faeces and the cecal microbiota ofdomestic and wild turkeys (A4, A5). However, little is known about thepathogenic potential of Sutterella sp. According to the Sutterellasp.-specific PCR methods described herein, Sutterella detection can beachieved in a mammal, such as a dog, a cat, a cow, a horse, a rabbit, amonkey, a pig, a sheep, a goat, a turkey, or a human.

Sutterella bacterial infections have been associated with ASD inaddition to Crohn's disease and ulcerative colitis. Bacterial infectionsare also associated with various intestinal diseases, such as irritablebowel syndrome (IBS). Over 40 million people in the U.S. suffer fromirritable bowel syndrome (IBS), a type of inflammatory bowel disease.IBS, though not fatal, has a huge impact on quality-of-life. After thecommon cold, IBS is the second most common reason for missed work and isestimated to generate $30B in healthcare costs. Few simple moleculardiagnostic tests for IBS/IBD are presently available. Diagnosis usuallyrelies upon symptom analysis and/or invasive colonoscopy procedures. TheIBD/IBS diagnostics market has significant growth potential.

Little is known of the epidemiology and pathogenesis of Sutterellainfection and their role in Crohn's disease, ASD, and ulcerativecolitis. Current methods for Sutterella biopsies are costly, laboriousand non-specific. There are no known rapid, specific, or cost-effectivetechnologies to identify Sutterella sp. in biological samples.

An aspect of the invention provides for a PCR assay that allows forrapid identification, quantification, classification, and diagnosis ofSutterella sp. in biological or industrial samples. This would allow forspecific therapies to be implemented in subjects in need (e.g., ASDpatients, IB patients, intestinal disease patients, etc.) followingidentification of Sutterella in infections. Directed administration ofantimicrobial agents (e.g., antibiotics) that limit the growth ofSutterella could be fascilitated rapidly following identification ofSutterella species. An antibiotic refers to any compound known to one ofordinary skill in the art that will inhibit the growth of, or kill,bacteria. Useful, non-limiting examples of an antibiotic includelincosamides (clindomycin); chloramphenicols; tetracyclines (such asTetracycline, Chlortetracycline, Demeclocycline, Methacycline,Doxycycline, Minocycline); aminoglycosides (such as Gentamicin,Tobramycin, Netilmicin, Amikacin, Kanamycin, Streptomycin, Neomycin);beta-lactams (such as penicillins, cephalosporins, Imipenem, Aztreonam);vancomycins; bacitracins; macrolides (erythromycins), amphotericins;sulfonamides (such as Sulfanilamide, Sulfamethoxazole, Sulfacetamide,Sulfadiazine, Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide,p-Aminobenzoic Acid, Trimethoprim-Sulfamethoxazole); Methenamin;Nitrofurantoin; Phenazopyridine; trimethoprim; rifampicins;metronidazoles; cefazolins; Lincomycin; Spectinomycin; mupirocins;quinolones (such as Nalidixic Acid, Cinoxacin, Norfloxacin,Ciprofloxacin, Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin,Levofloxacin); novobiocins; polymixins; gramicidins; andantipseudomonals (such as Carbenicillin, Carbenicillin Indanyl,Ticarcillin, Azlocillin, Mezlocillin, Piperacillin) or any salts orvariants thereof. Such antibiotics can be obtained commercially, e.g.,from Daiichi Sankyo, Inc. (Parsipanny, N.J.), Merck (Whitehouse Station,N.J.), Pfizer (New York, N.Y.), Glaxo Smith Kline (Research TrianglePark, N.C.), Johnson & Johnson (New Brunswick, N.J.), AstraZeneca(Wilmington, Del.), Novartis (East Hanover, N.J.), and Sanofi-Aventis(Bridgewater, N.J.). The antibiotic used will depend on the type ofbacterial infection.

In one embodiment, the invention provides for a method of detectingSutterella sp. DNA from biological or industrial sources, e.g.intestinal tissue, feces, blood, or skin. In another embodiment, theinvention provides for Sutterella diagnostics to detect Sutterella sp.in samples from children with autism as well as patients with intestinaldisease, e.g. irritable bowel syndrome (IBS). In some embodiments, theinvention provides for PCR-based methods of assessing a subject'sresponse to exposure to therapeutic treatments directed at bacterialinfections, for example, Sutterella sp. infections, or exposure to otherpathogens.

For example, primers having SEQ ID NOS: 11, 12, 15, or 16 can be usedfor detecting Sutterella sp. DNA. SEQ ID NOS: 17 and 18 can also be usedfor detecting Sutterella sp. DNA.

Sutt For Primer (SEQ ID NO: 17)-

TTGACCATG

C

GAA

BB

-3′ Sutt Rev Primer (SEQ ID NO: 18)- 5′-CCCTCTGTTCCGACCATTGTATGACGTGTGA

GCCC

AGCC

TAAGGGCCA TGAGGACT-3′ Sutt Probe 3 (SEQ ID NO: 19)-

TGTCGTG

GAT

T

TTA

GTCCCGCA

C

AGCGCAACCCT

G

CA

-3′

In addition to the primers having SEQ ID NOS: 11, 12, and 15-18,additional primers containing any part of SEQ ID NOS: 17, 18, or 19 andcontaining any portion of the italicized DNA sequence regions can beused to assess the presence or absence of Sutterella species. Further,inclusion of degenerate bases (bolded and underlined) can be used toincrease coverage of Sutterella species (for example, where S can be a Gnucleotide and/or a C nucleotide; where Y can be a C nucleotide and/or Tnucleotide; where R can be an A nucleotide and/or G nucleotide; where Wcan be an A nucleotide and/or T nucleotide; where H can be an Anucleotide and/or T nucleotide and/or C nucleotide; where B can be a Tnucleotide, C nucleotide, or G nucleotide; where V can be an Anucleotide, G nucleotide, or C nucleotide; where D can be an Anucleotide, G nucleotide, or T nucleotide; where K can be a G nucleotideor T nucleotide).

In addition to the highlighted probe sequence of SEQ ID NO:19 as well asSEQ ID NOS: 13 and 14, any portion of SEQ ID NO: 19 shown above can beused for Sutterella species detection and quantitation. The reversecomplement of SEQ ID NOS: 11, 12, or 15-19 can also be used to detectthe opposite DNA strand of Sutterella species 16S rRNA genes.

The invention can be used to detect Sutterella sp. 16S rRNA sequences insmall amounts of DNA from any biological or industrial source. Thesesources include, but are not limited to human or animal intestinaltissue, feces, blood, or skin (swabs or tissue). Based on thesefindings, the invention can be used to detect, quantitate, and classifySutterella sp. in biological samples from children with Autism. In oneembodiment, the invention can be used to detect Sutterella sp. inbiological samples from individuals with various forms of intestinaldisease. Intestinal diseases include, but are not limited to, Crohn'sdisease and Ulcerative colitis. In one embodiment, detection ofSutterella sp. can occur in biological samples from any undiagnosedinfection below or above the diaphragm. The invention will allow forlarge cohort investigations of Sutterella sp. in the aforementioned, andas yet to be determined, diseases in order to establish an associationbetween Sutterella sp. and disease manifestation. In one embodiment, thepresence and quantity of Sutterella sp. in intestinal tissues can beinvestigated following any number of experimental manipulations.Experimental manipulations include, but are not limited to, responses tochemicals (i.e. antibiotics), changes in diet, pathogen exposure (i.e.pathogenic viruses, bacteria, fungi), or probiotic usage. The rapididentification of Sutterella sp. in human and animal samples facilitatedby this invention can lead to rapid diagnosis and directed antimicrobialtreatment of infections caused by Sutterella sp.

Gene. Vectors, Recombinant Cells, and Polypeptides

The invention encompasses an altered or mutated genes of a carbohydratetransporter or carbohydrate metabolic enzyme, or a fragment thereof. Theinvention also encompasses nucleic acid molecules encoding an altered ormutated polypeptide of s carbohydrate transporter or carbohydratemetabolic enzyme, or a fragment thereof. The alteration or mutation ofthe nucleotide or amino acid sequence modifies the carbohydratetransporter or carbohydrate metabolic enzyme activity, respectively. Theinvention provides for a vector that comprises a nucleic acid encoding acarbohydrate transporter or carbohydrate metabolic enzyme polypeptide(for example, a nucleic acid comprising SEQ ID NO: 2 or 4, and a nucleicacid comprising SEQ ID NO: 6, 8, or 10, respectively) or mutant thereof.The vector can be a cloning vector or an expression vector, i.e., avector comprising regulatory sequences causing resulting in theexpression of carbohydrate transporter or carbohydrate metabolic enzymepolypeptides from the vector in a competent host cell. These vectors canbe used to express polypeptides, or mutants thereof, of carbohydratetransporters or carbohydrate metabolic enzymes in vitro, ex vivo, or invivo, to create transgenic or Knock-Out non-human animals, to amplifythe nucleic acids, or to express antisense RNAs.

The nucleic acids used to practice the invention, whether RNA, RNAi,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, can be produced or isolated from a variety of sources,genetically engineered, amplified, and/or expressed/generatedrecombinantly. Recombinant polypeptides generated from these nucleicacids can be individually isolated or cloned and tested for a desiredactivity. Any recombinant expression system can be used, includingbacterial, mammalian, yeast, insect or plant cell expression systems.Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams,J. Am. Chem. Soc. 105:661, 1983; Belousov, Nucleic Acids Res.25:3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19:373-380, 1995;Blommers, Biochemistry 33:7886-7896, 1994; Narang, Meth. Enzymol. 68:90,1979; Brown Meth. Enzymol. 68:109, 1979; Beaucage, Tetra. Lett. 22:1859,1981; U.S. Pat. No. 4,458,066, all of which are incorporated byreference in their entireties.

The invention provides oligonucleotides comprising sequences of theinvention, e.g., subsequences of the exemplary sequences of theinvention. Oligonucleotides can include, e.g., single strandedpoly-deoxynucleotides or two complementary polydeoxynucleotide strandswhich can be chemically synthesized.

Techniques for the manipulation of nucleic acids, such as, subcloning,labeling probes (for example, random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, andhybridization are well described in the scientific and patentliterature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORYMANUAL (2^(ND) ED.), Vols. 1-3, Cold Spring Harbor Laboratory, 1989;CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons,Inc., New York, 1997; LABORATORY TECHNIQUES IN BIOCHEMISTRY ANDMOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I.Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.

Nucleic acids, vectors, or polypeptides can be analyzed and quantifiedby any of a number of general means well known to those of skill in theart. These include, for example, analytical biochemical methods such asradiography, electrophoresis, NMR, spectrophotometry, capillaryelectrophoresis, thin layer chromatography (TLC), high performanceliquid chromatography (HPLC), and hyperdiffusion chromatography; variousimmunological methods, such as immuno-electrophoresis, Southernanalysis, Northern analysis, dot-blot analysis, fluid or gelprecipitation reactions, immunodiffusion, quadrature radioimmunoassay(RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescentassays, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target orsignal amplification methods, radiolabeling, scintillation counting, andaffinity chromatography.

Obtaining and manipulating nucleic acids used to practice the methods ofthe invention can be done by cloning from genomic samples, and, ifdesired, screening and re-cloning inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g.,Rosenfeld, Nat. Genet. 15:333-335, 1997; yeast artificial chromosomes(YAC); bacterial artificial chromosomes (BAC); P1 artificialchromosomes, see, e.g., Woon, Genomics 50:306-316, 1998; P1-derivedvectors (PACs), see, e.g., Kern, Biotechniques 23:120-124, 1997;cosmids, recombinant viruses, phages or plasmids

The vectors of this invention can comprise a coding sequence for acarbohydrate transporter molecule or a carbohydrate metabolic enzymemolecule that is operably linked to regulatory sequences, e.g., apromoter, or a polyA tail. Operably linked indicates that the coding andregulatory sequences are functionally associated so that the regulatorysequences cause expression (e.g., transcription) of the codingsequences. The vectors can further comprise one or several origins ofreplication and/or selectable markers. The promoter region can behomologous or heterologous with respect to the coding sequence, and canprovide for ubiquitous, constitutive, regulated and/or tissue specificexpression, in any appropriate host cell, including for in vivo use.Examples of promoters include bacterial promoters (T7, pTAC, Trppromoter), viral promoters (LTR, TK, CMV-IE), mammalian gene promoters(albumin, PGK), etc.

The vector can be a plasmid, a virus, a cosmid, a phage, a BAC, a YAC.Plasmid vectors can be prepared from commercially available vectors suchas pBluescript, pUC, or pBR. Viral vectors can be produced frombaculoviruses, retroviruses, adenoviruses, or AAVs, according torecombinant DNA techniques known in the art. In one embodiment, arecombinant virus can encode a polypeptide of a carbohydrate transporteror carbohydrate metabolic enzyme of the invention. The recombinant virusis useful if replication-defective, for example, if selected from E1-and/or E4-defective adenoviruses, Gag-, pol- and/or env-defectiveretroviruses and Rep- and/or Cap-defective AAVs. Such recombinantviruses can be produced by techniques known in the art, such as bytransfecting packaging cells or by transient transfection with helperplasmids or viruses. Examples of virus packaging cells include PA317cells, PsiCRIP cells, GPenv+ cells, or 293 cells. Detailed protocols forproducing such replication-defective recombinant viruses can be foundfor instance in WO95/14785, WO96/22378, U.S. Pat. Nos. 5,882,877,6,013,516, 4,861,719, 5,278,056 and WO94/19478, which are all herebyincorporated by reference.

In another embodiment, the invention provides for a recombinant hostcell comprising a recombinant carbohydrate transporter gene (e.g., GLUT2or SGLT1) or a carbohydrate metabolic enzyme gene (e.g., SI, MGAM, orLCT), or a recombinant vector as described herein. Suitable host cellsinclude, without limitation, prokaryotic cells (such as bacteria) andeukaryotic cells (such as yeast cells, mammalian cells, insect cells, orplant cells). Specific examples include E. coli, the yeastsKluyveromyces or Saccharomyces, mammalian cell lines (e.g., Vero cells,CHO cells, 3T3 cells, or COS cells) as well as primary or establishedmammalian cell cultures (e.g., produced from fibroblasts, embryoniccells, epithelial cells, nervous cells, or adipocytes). In a furtherembodiment, the invention provides a method for producing a recombinanthost cell expressing a polypeptide of a carbohydrate transporter orcarbohydrate metabolic enzyme. The method can entail (a) introducing invitro or ex vivo into a competent host cell a recombinant nucleic acidor a vector as described herein, (b) culturing in vitro or ex vivo therecombinant host cells obtained, and (c) optionally, selecting the cellswhich express the polypeptide of a carbohydrate transporter orcarbohydrate metabolic enzyme. Such recombinant host cells can be usedfor the production of carbohydrate transporter or carbohydrate metabolicenzyme polypeptides, as well as for screening of active molecules, asdescribed below. Such cells can also be used as a model system to studyautism. These cells can be maintained in suitable culture media, such asHAM, DMEM, or RPMI, in any appropriate culture device (plate, flask,dish, tube, or pouch).

The practice of aspects of the present invention can employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Caner and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).All patents, patent applications and references cited herein areincorporated in their entirety by reference.

Administration and Dosing

A carbohydrate transporter molecule (e.g., GLUT2 or SGLT1) orcarbohydrate metabolic enzyme molecule (e.g., SI, MGAM, or LCT) can beadministered to the subject once (e.g., as a single injection ordeposition). Alternatively, a carbohydrate transporter or carbohydratemetabolic enzyme molecule of the invention can be administered once ortwice daily to a subject in need thereof for a period of from about twoto about twenty-eight days, or from about seven to about ten days. Itcan also be administered once or twice daily to a subject for a periodof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or acombination thereof. Furthermore, the carbohydrate transporter orcarbohydrate metabolic enzyme molecule of the invention can beco-administrated with another therapeutic, such as an anti-depressant,an anti-psychotic, a benzodiazepine drug, or a combination thereof.Where a dosage regimen comprises multiple administrations, the effectiveamount of the carbohydrate transporter or carbohydrate metabolic enzymemolecule administered to the subject can comprise the total amount ofgene product administered over the entire dosage regimen.

The carbohydrate transporter or carbohydrate metabolic enzyme moleculesof the invention can be administered to a subject by any means suitablefor delivering the carbohydrate transporter or carbohydrate metabolicenzyme molecules to cells of the subject, such as ileum cell or cecumcells. For example, carbohydrate transporter or carbohydrate metabolicenzyme molecules can be administered by methods suitable to transfectcells. Transfection methods for eukaryotic cells are well known in theart, and include direct injection of the nucleic acid into the nucleusor pronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

The compositions of this invention can be formulated and administered toreduce the symptoms associated with autism or an ASD by any means thatproduces contact of the active ingredient with the agent's site ofaction in the body of an animal. They can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic active ingredients orin a combination of therapeutic active ingredients. They can beadministered alone, but are generally administered with a pharmaceuticalcarrier selected on the basis of the chosen route of administration andstandard pharmaceutical practice.

Pharmaceutical compositions for use in accordance with the invention canbe formulated in conventional manner using one or more physiologicallyacceptable carriers or excipients. The therapeutic compositions of theinvention can be formulated for a variety of routes of administration,including systemic and topical or localized administration. Techniquesand formulations generally can be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. (1985), the entiredisclosure of which is herein incorporated by reference. For systemicadministration, an injection is useful, including intramuscular,intravenous, intraperitoneal, and subcutaneous. For injection, thetherapeutic compositions of the invention can be formulated in liquidsolutions, for example in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the therapeuticcompositions can be formulated in solid form and redissolved orsuspended immediately prior to use.

Lyophilized forms are also included. Pharmaceutical compositions of thepresent invention are characterized as being at least sterile andpyrogen-free. These pharmaceutical formulations include formulations forhuman and veterinary use.

Pharmaceutical formulations of the invention can comprise a carbohydratetransporter or carbohydrate metabolic enzyme molecule (e.g., 0.1 to 90%by weight), or a physiologically acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. The pharmaceutical formulations ofthe invention can also comprise the carbohydrate transporter orcarbohydrate metabolic enzyme molecules of the invention which areencapsulated by liposomes and a pharmaceutically-acceptable carrier.Useful pharmaceutically-acceptable carriers are water, buffered water,normal saline, 0.4% saline, 0.3% glycine, or hyaluronic acid.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose, ormagnesium carbonate.

Solid formulations can be used for enteral (oral) administration. Theycan be formulated as, e.g., pills, tablets, powders or capsules. Forsolid compositions, conventional nontoxic solid carriers can be usedwhich include, e.g., pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, or magnesium carbonate. For oral administration, apharmaceutically acceptable nontoxic composition is formed byincorporating any of the normally employed excipients, such as thosecarriers previously listed, and generally 10% to 95% of activeingredient (e.g., peptide). A non-solid formulation can also be used forenteral administration. The carrier can be selected from various oilsincluding those of petroleum, animal, vegetable or synthetic origin,e.g., peanut oil, soybean oil, mineral oil, or sesame oil. Suitablepharmaceutical excipients include e.g., starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk, glycerol, propylene glycol, water, ethanol.

Nucleic acids, peptides, or polypeptides of the invention, whenadministered orally, can be protected from digestion. This can beaccomplished either by complexing the nucleic acid, peptide orpolypeptide with a composition to render it resistant to acidic andenzymatic hydrolysis or by packaging the nucleic acid, peptide orpolypeptide in an appropriately resistant carrier such as a liposome.Means of protecting compounds from digestion are well known in the art,see, e.g., Fix, Pharm Res. 13: 1760-1764, 1996; Samanen, J. Pharm.Pharmacol. 48: 119-135, 1996; U.S. Pat. No. 5,391,377, describing lipidcompositions for oral delivery of therapeutic agents (for example,liposomal delivery). In one embodiment, the carbohydrate transportermolecule (e.g., GLUT2 or SGLT1) or carbohydrate metabolic enzymemolecule (e.g., SI, MGAM, or LCT) can be delivered to the alimentarycanal or intestine of the subject via oral administration that is canwithstand digestion and degradation.

For oral administration, the therapeutic compositions can take the formof, for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active agent. For buccal administration thetherapeutic compositions can take the form of tablets or lozengesformulated in a conventional manner. For administration by inhalation,the compositions for use according to the present invention areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflate or can beformulated containing a powder mix of the therapeutic agents and asuitable powder base such as lactose or starch.

The therapeutic compositions can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection can be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Suitable enteral administration routes for the present methods includeoral, rectal, or intranasal delivery. Suitable parenteral administrationroutes include intravascular administration (e.g. intravenous bolusinjection, intravenous infusion, intra-arterial bolus injection,intra-arterial infusion and catheter instillation into the vasculature);peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoralinjection, intra-retinal injection, or subretinal injection);subcutaneous injection or deposition including subcutaneous infusion(such as by osmotic pumps); direct application to the tissue ofinterest, for example by a catheter or other placement device (e.g., aretinal pellet or a suppository or an implant comprising a porous,non-porous, or gelatinous material); and inhalation. For example, thecarbohydrate transporter or carbohydrate metabolic enzyme molecules ofthe invention can be administered by injection, infusion, or oraldelivery.

In addition to the formulations described previously, the therapeuticcompositions can also be formulated as a depot preparation. Such longacting formulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Forexample, the therapeutic compositions can be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. In addition, detergents can be used to facilitatepermeation. Transmucosal administration can be through nasal sprays orusing suppositories. For topical administration, the compositions of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing. For oraladministration, the therapeutic compositions are formulated intoconventional oral administration forms such as capsules, tablets, andtonics.

A composition of the present invention can also be formulated as asustained and/or timed release formulation. Such sustained and/or timedrelease formulations can be made by sustained release means or deliverydevices that are well known to those of ordinary skill in the art, suchas those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, thedisclosures of which are each incorporated herein by reference. Thepharmaceutical compositions of the present invention can be used toprovide slow or sustained release of one or more of the activeingredients using, for example, hydropropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or the like, or acombination thereof to provide the desired release profile in varyingproportions. Suitable sustained release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the pharmaceutical compositions of theinvention. Single unit dosage forms suitable for oral administration,such as, but not limited to, tablets, capsules, gel-caps, caplets, orpowders, that are adapted for sustained release are encompassed by thepresent invention.

In the present methods, the carbohydrate transporter or carbohydratemetabolic enzyme molecules can be administered to the subject either asRNA, in conjunction with a delivery reagent, or as a nucleic acid (e.g.,a recombinant plasmid or viral vector) comprising sequences whichexpresses the gene product. Suitable delivery reagents foradministration of the carbohydrate transporter or carbohydrate metabolicenzyme molecules include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes.

The dosage administered can be a therapeutically effective amount of thecomposition sufficient to result in amelioration of symptoms of autismor an autism spectrum disorder in a subject, and can vary depending uponknown factors such as the pharmacodynamic characteristics of the activeingredient and its mode and route of administration; age, sex, healthand weight of the recipient; nature and extent of symptoms; kind ofconcurrent treatment, frequency of treatment and the effect desired. Forexample, an effective enzyme unit of amount of SI, MGAM, and/or LCT canbe administered to a subject in need thereof. The enzyme unit (U) is aunit for the amount of a particular enzyme. One U is defined as theamount of the enzyme that catalyzes the conversion of 1 micro mole ofsubstrate per minute. In one embodiment, the therapeutically effectiveamount of the administered carbohydrate enzyme (e.g., SI, MGAM, or LCT)is at least about 1 U, at least about 10 U, at least about 20 U, atleast about 25 U, at least about 50 U, at least about 100 U, at leastabout 150 U, at least about 200 U, at least about 250 U, at least about300 U, at least about 350 U, at least about 400 U, at least about 450 U,at least about 500 U, at least about 550 U, at least about 600 U, atleast about 650 U, at least about 700 U, at least about 750 U, at leastabout 800 U, at least about 850 U, at least about 900 U, at least about950 U, at least about 1000 U, at least about 1250 U, at least about 1500U, at least about 1750 U, at least about 2000 U, at least about 2250 U,at least about 2500 U, at least about 2750 U, at least about 3000 U, atleast about 3250 U, at least about 3500 U, at least about 4000 U, atleast about 4500 U, at least about 5000 U, at least about 5500 U, atleast about 6000 U, at least about 6500 U, at least about 7000 U, atleast about 7500 U, at least about 8000 U, at least about 8500 U, atleast about 9000 U, at least about 9250 U, at least about 9500 U, or atleast about 10,000 U.

In some embodiments, the effective amount of the administeredcarboydrate transporter molecule (e.g., GLUT2 or SGLT1) is at leastabout 0.01 μg/kg body weight, at least about 0.025 μg/kg body weight, atleast about 0.05 μg/kg body weight, at least about 0.075 μg/kg bodyweight, at least about 0.1 μg/kg body weight, at least about 0.25 μg/kgbody weight, at least about 0.5 μg/kg body weight, at least about 0.75μg/kg body weight, at least about 1 μg/kg body weight, at least about 5μg/kg body weight, at least about 10 μg/kg body weight, at least about25 μg/kg body weight, at least about 50 μg/kg body weight, at leastabout 75 μg/kg body weight, at least about 100 μg/kg body weight, atleast about 150 μg/kg body weight, at least about 200 μg/kg body weight,at least about 250 μg/kg body weight, at least about 300 μg/kg bodyweight, at least about 350 μg/kg body weight, at least about 400 μg/kgbody weight, at least about 450 μg/kg body weight, at least about 500μg/kg body weight, at least about 550 μg/kg body weight, at least about600 μg/kg body weight, at least about 650 μg/kg body weight, at leastabout 700 μg/kg body weight, at least about 750 μg/kg body weight, atleast about 800 μg/kg body weight, at least about 850 μg/kg body weight,at least about 900 μg/kg body weight, at least about 950 μg/kg bodyweight, or at least about 1000 μg/kg body weight.

Toxicity and therapeutic efficacy of therapeutic compositions of thepresent invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Therapeutic agentsthat exhibit large therapeutic indices are useful. Therapeuticcompositions that exhibit some toxic side effects can be used.

A therapeutically effective dose of carbohydrate transporter orcarbohydrate metabolic enzyme molecules can depend upon a number offactors known to those or ordinary skill in the art. The dose(s) of thecarbohydrate transporter or carbohydrate metabolic enzyme molecules canvary, for example, depending upon the identity, size, and condition ofthe subject or sample being treated, further depending upon the route bywhich the composition is to be administered, if applicable, and theeffect which the practitioner desires the carbohydrate transporter orcarbohydrate metabolic enzyme molecules to have upon the nucleic acid orpolypeptide of the invention. These amounts can be readily determined bya skilled artisan.

Pharmaceutical Composition and Therapy

The invention provides methods for treating or preventing autism or anautism spectrum disorder in a subject. In one embodiment, the method cancomprise administering to the subject a functional (e.g., wild-type)carbohydrate transporter molecule (e.g., GLUT2 or SGLT1) or carbohydratemetabolic enzyme molecule (e.g., SI, MGAM, or LCT), which can be apolypeptide or a nucleic acid.

Various approaches can be carried out to restore the carbohydratetransporter or carbohydrate metabolic enzyme activity or function in asubject, such as those carrying an altered gene locus comprising acarbohydrate transporter gene (e.g., GLUT2 or SGLT1) or a carbohydratemetabolic enzyme gene (e.g., SI, MGAM, or LCT). Supplying wild-typefunction of the carbohydrate transporter or carbohydrate metabolicenzyme to such subjects can suppress phenotypic expression of autism oran autism spectrum disorders in a pathological cell or organism.Increasing carbohydrate transporter or carbohydrate metabolic enzymeactivity can be accomplished through gene or protein therapy asdiscussed later herein.

A nucleic acid encoding a carbohydrate transporter or carbohydratemetabolic enzyme or a functional part thereof can be introduced into thecells of a subject in one embodiment of the invention. The wild-typecarbohydrate transporter gene or carbohydrate metabolic enzyme gene (ora functional part thereof) can also be introduced into the cells of thesubject in need thereof using a vector as described herein. The vectorcan be a viral vector or a plasmid. The gene can also be introduced asnaked DNA. The gene can be provided so as to integrate into the genomeof the recipient host cells, or to remain extra-chromosomal. Integrationcan occur randomly or at precisely defined sites, such as throughhomologous recombination. For example, a functional copy of thecarbohydrate transporter gene or a carbohydrate metabolic enzyme genecan be inserted in replacement of an altered version in a cell, throughhomologous recombination. Further techniques include gene gun,liposome-mediated transfection, or cationic lipid-mediated transfection.Gene therapy can be accomplished by direct gene injection, or byadministering ex vivo prepared genetically modified cells expressing afunctional polypeptide.

Gene Therapy and Protein Replacement Methods

Delivery of nucleic acids into viable cells can be effected ex vivo, insitu, or in vivo by use of vectors, and more particularly viral vectors(e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus),or ex vivo by use of physical DNA transfer methods (e.g., liposomes orchemical treatments). Non-limiting techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,and the calcium phosphate precipitation method (see, for example,Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)).Introduction of a nucleic acid or a gene encoding a polypeptide of theinvention can also be accomplished with extrachromosomal substrates(transient expression) or artificial chromosomes (stable expression).Cells can also be cultured ex vivo in the presence of therapeuticcompositions of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapyvectors. A number of viruses have been used as gene transfer vectors,including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus(Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992;Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992;Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992),adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992;Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990),and retroviruses of avian (Biandyopadhyay and Temin, 1984; Petropouloset al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al.,1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin(Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990;Buchschacher and Panganiban, 1992). Non-limiting examples of in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporatedby reference in its entirety) and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993),incorporated entirely by reference). For example, naked DNA vaccines aregenerally known in the art; see Brower, Nature Biotechnology,16:1304-1305 (1998), which is incorporated by reference in its entirety.Gene therapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see, e.g., U.S. Pat. No.5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994.Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g., retroviral vectors, the pharmaceutical preparation caninclude one or more cells that produce the gene delivery system.

For reviews of gene therapy protocols and methods see Anderson et al.,Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469,6,017,524, 6,143,290, 6,410,010 6,511,847; and U.S. ApplicationPublication Nos. 2002/0077313 and 2002/00069, which are all herebyincorporated by reference in their entireties. For additional reviews ofgene therapy technology, see Friedmann, Science, 244:1275-1281 (1989);Verma, Scientific American: 68-84 (1990); Miller, Nature, 357: 455-460(1992); Kikuchi et al., J Dermatol Sci. 2008 May; 50(2):87-98; Isaka etal., Expert Opin Drug Deliv. 2007 September; 4(5):561-71; Jager et al.,Curr Gene Ther. 2007 August; 7(4):272-83; Waehler et al., Nat Rev Genet.2007 August; 8(8):573-87; Jensen et al., Ann Med. 2007; 39(2):108-15;Herweijer et al., Gene Ther. 2007 January; 14(2):99-107; Eliyahu et al.,Molecules, 2005 Jan. 31; 10(1):34-64; and Altaras et al., Adv BiochemEng Biotechnol. 2005; 99:193-260, all of which are hereby incorporatedby reference in their entireties.

Protein replacement therapy can increase the amount of protein byexogenously introducing wild-type or biologically functional protein byway of infusion. A replacement polypeptide can be synthesized accordingto known chemical techniques or can be produced and purified via knownmolecular biological techniques. Protein replacement therapy has beendeveloped for various disorders. For example, a wild-type protein can bepurified from a recombinant cellular expression system (e.g., mammaliancells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.;U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No.6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et al.;U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No. 6,451,600 toRasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen et al. and U.S.Pat. No. 5,879,680 to Ginns et al.), human placenta, or animal milk (seeU.S. Pat. No. 6,188,045 to Reuser et al.), or other sources known in theart. After the infusion, the exogenous protein can be taken up bytissues through non-specific or receptor-mediated mechanism.

A polypeptide encoded by a carbohydrate transporter gene (e.g., GLUT2 orSGLT1) or a carbohydrate metabolic enzyme gene (for example, SI, MGAM,or LCT) can also be delivered in a controlled release system. Forexample, the polypeptide can be administered using intravenous infusion,an implantable osmotic pump, a transdermal patch, liposomes, or othermodes of administration. In one embodiment, a pump can be used (see isLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application is understood by theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

* * *

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 1 Identification of Carbohydrate Transporters and CarbohydrateMetabolic Enzymes as Biomarkers in a Subset of Autism Spectrum Disorders(ASD)

Gastrointestinal disturbances complicate clinical management in somechildren with autism. Reports of ileo-colonic lymphoid nodularhyperplasia and deficiencies in disaccharidase enzymatic activity led tothe survey of intestinal gene expression and microflora in children withautism and gastrointestinal disease (AUT-GI) or gastrointestinal diseasealone (Control-GI). In AUT-GI subjects, ileal transcripts for thedisaccharidases sucrase isomaltase, maltase glucoamylase, and lactase,and the monosaccharide transporters, sodium-dependent glucoseco-transporter, and glucose transporter 2 were significantly decreased.Alterations in intestinal carbohydrates as a result of thesedeficiencies would have a distinct impact on the composition of AUT-GIintestinal microbiota. Bacterial 16S rRNA gene pyrosequencing analysisof biopsy material from ileum and cecum revealed decreasedBacteroidetes, increased Firmicute/Bacteroidete ratios, highercumulative levels of Firmicutes and Proteobacteria, and increasedBetaproteobacteria in AUT-GI as compared with Control-GI biopsies. Theseresults indicate a complex dependence between intestinal gene expressionand bacterial community structure that contributes to gastrointestinaldysfunction in AUT-GI children.

Deficiencies in intestinal disaccharidase and/or glucoamylase activityare reported in over half of autistic children with gastrointestinaldisturbances (AUT-GI) (Horvath et al., 1999). To determine whetherfunctional deficits reflect decreased levels of mRNA encoding theseenzymes transcript levels were examined for three primary brush borderdisaccharidases (sucrase isomaltase [SI], maltase glucoamylase [MGAM],and lactase [LCT]) in ileal biopsies of AUT-GI and Control-GI childrenby real time PCR. Levels of mRNA for all three enzymes were decreased inAUT-GI: SI (FIG. 16A: Mann-Whitney, p=0.001), MGAM (FIG. 16B:Mann-Whitney, p=0.003) and LCT (FIG. 16C: Mann-Whitney, p=0.032).Deficiencies in LCT mRNA in AUT-GI children were not attributable todisproportionate adult-type hypolactasia genotypes in the AUT-GI grouprelative to the Control-GI group (FIGS. 21A-21E and Methods). Within theASD-GI group, 86.7% (SI), 80% (MGAM), and 80% (LCT) of children hadtranscript levels below the 25^(th) percentile of Control-GI children(Table 5A). Nearly all (14/15, or 93.3%) AUT-GI children haddeficiencies in at least one disaccharidase enzyme; 80% had deficienciesin 2 or more enzymes; and 73.3% had deficiencies in all three enzymes(Table 5A). Tables 5A-C are summary tables for gene expression andbacterial assays. Increases or decreases in AUT-GI children in both geneexpression and bacterial parameters were determined for each individualbased on the levels of each parameter in the Control-GI group. Thevalues for a given parameter in the AUT-GI children that exceeded the75^(th) (arrow pointing up) percentile or were below the 25^(th)percentile (arrow pointing down) for the corresponding parameter in theControl-GI children were scored as an increase or decrease,respectively. Values that were also above the 90^(th) or below the10^(th) percentiles of Control-GI children are indicated by doublearrows.

TABLE 5A Summary tables for gene expression and bacterial assays. ASDPatient # SI MGAM LCT SGLT1 GLUT2 CDX2 Villin # Disaccharidases #Transporters Total 1

 

 

 

 

 

n.c. 3/3 2/2 5/5 2

 

 

 

 

 

 

 

3/3 2/2 5/5 3

 

 

 

 

 

 

 

3/3 2/2 5/5 4

 

 

 

 

 

n.c.

 

3/3 2/2 5/5 5

 

 

 

 

 

 

 

3/3 2/2 5/5 6

 

n.c. n.c. n.c. n.c. n.c. n.c. 1/3 0/2 1/5 7 n.c. n.c.

 

n.c. n.c.

 

0/3 0/2 0/5 8

 

 

 

 

n.c.

3/3 2/2 5/5 9

 

 

 

 

n.c. n.c.

3/3 1/2 4/5 10

 

 

 

 

 

n.c.

3/3 212 5/5 11

 

 

 

 

 

n.c.

3/3 2/2 5/5 12

 

 

 

 

 

n.c. n.c. 3/3 2/2 5/5 13

 

 

 

 

 

 

 

3/3 2/2 5/5 14

 

 

n.c. n.c.

 

n.c.

 

2/3 1/2 3/5 15 n.c. n.c.

 

n.c. n.c. n.c. n.c. 1/3 0/2 1/5 % below 86.7% 80.0% 80.0% 73.3% 73.3%33.3% 26.7% Summary Summary Summary controls All 3 = 73.3% Both = 66.7%All 5 = 66.7% At least 2 = 80% At least 1 = 80% At least 4 = 73.3% Atleast 1 = 93.3% At least 3 = 80% At least 1 = 93.3%

TABLE 5B Summary tables for gene expression and bacterial assays.

TABLE 5C Summary tables for gene expression and bacterial assays. Firm./Firm./ Firm./ Firm./ Clostridiales/ Clostridiales/ Firm. + Firm. +Bacteroid. Bacteroid. Bacteroid. Bacteroid. Bacteroidales BacteroidalesProteobac. Proteobac. ASD Ratio—RT Ratio—RT Ratio—454 Ratio—454Ratio—454 Ratio—454 Ratio—454 Ratio—454 Patient # Ileum Cecum IleumCecum Ileum Cecum Ileum Ileum 1

 

 

 

 

 

 

 

 

2

 

 

 

 

 

 

3

 

 

 

 

 

 

4

 

 

 

 

 

 

 

 

5

 

n.c.

n.c.

n.c.

n.c. 6

 

 

n.c. n.c.

n.c. n.c.

7

 

 

 

 

 

 

8

 

 

 

 

 

 

 

9

 

n.c.

 

 

 

 

 

 

10

n.c.

 

 

 

 

 

 

11

 

 

 

 

 

12

 

n.c. n.c. n.c. n.c. n.c. n.c. n.c. 13

 

 

 

 

 

 

 

14

n.c.

 

n.c.

 

n.c.

n.c. 15

 

 

 

 

 

% above 100% 60% 73.3% 66.7% 80.0% 66.7% 80% 73.3% controls

Two hexose transporters, SGLT1 and GLUT2, mediate transport ofmonosaccharides in the intestine. SGLT1, located on the luminal membraneof enterocytes, is responsible for the active transport of glucose andgalactose from the intestinal lumen into enterocytes. GLUT2 transportsglucose, galactose and fructose across the basolateral membrane into thecirculation and can also translocate to the apical membrane (Kellett etal., 2008). Real-time PCR revealed a decrease in SGLT1 mRNA (FIG. 16D:Mann-Whitney, p=0.008) and GLUT2 mRNA (FIG. 16E: Mann-Whitney, p=0.010)in AUT-GI children. For SGLT1, 73.3% of AUT-GI children had transcriptlevels below the 25th percentile of Control-GI children; 73.3% of AUT-GIchildren had GLUT2 transcript levels below the 25^(th) percentile ofControl-GI children (Table 5A). Deficiencies were found in at least onehexose transporter in 80% of AUT-GI children; 66.7% had deficiencies inboth transporters. In total, 66.7% of AUT-GI children had mRNAdeficiencies in all 5 molecules involved in carbohydrate digestion andtransport (Table 5A). Expression levels were correlated(Bonferroni-adjusted Spearman rank order correlations) in the AUT-GIgroup for all gene combinations except LCT and GLUT2, for which only atrend was observed. In the Control-GI group, significance was limited tocorrelations of SI-MGAM, MGAM-SGLT1, and LCT-SGLT1 (Table 2).

TABLE 2 Spearman correlations between ileal gene expression andbacterial abundance variables. Group SI MGAM LCT SGLT1 GLUT2 Villin CDX2SI AUT 1 0.89*** 0.59* 0.88** 0.76** 0.24 0.59* Control 1 0.93* 0.540.68† 0.75† 0.57 0.68† MGAM AUT — 1 0.56* 0.86** 0.75** 0.31 0.63*Control — 1 0.75† 0.82* 0.64 0.71† 0.82* LCT AUT — — 1 0.62* 0.52† 0.58*0.65* Control — — 1 0.86* 0.57 0.82* 0.86* SGLT1 AUT — — — 1 0.71** 0.340.54* Control — — — 1 0.64 0.96* 1.00* GLUT2 AUT — — — — 1 0.51† 0.69**Control — — — — 1 0.54 0.64 Villin AUT — — — — — 1 0.60* Control — — — —— 1 0.96* CDX2 AUT — — — — — — 1 Control — — — — — — 1 Bacteroidetes AUT0.33 0.10 0.31 0.52†^(a) 0.07 0.02 −0.01 Ileum Control −0.29 −0.29 −0.32−0.18 −0.75†^(a) 0.00 −0.18 Bacteroidetes AUT 0.18 0.06 0.23 0.33 0.050.12 0.10 Cecum Control −0.93* −1.00* −0.75† −0.82* −0.64 −0.71† −0.82*Firmicutes AUT −0.61*^(a) −0.55*^(a) −0.00 0.12 0.23 0.64* 0.48†^(a)Ileum Control 0.43 0.36 0.18 0.32 0.61 0.14 0.32 Firmicutes AUT −0.060.05 −0.05 0.04 0.15 0.58* 0.14 Cecum Control 0.86* 0.86* 0.68† 0.89*0.86* 0.79† 0.89* Firm./Bacteroid. AUT −0.72**^(a) −0.65*^(a) −0.61*^(a)−0.65*^(a) −0.55*^(a) 0.36 −0.58*^(a) Ileum Control 0.43 0.36 0.18 0.320.61 0.14 0.32 Firm./Bacteroid. AUT −0.51†^(a) −0.08 −0.11 −0.23 0.000.42 0.06 Cecum Control 0.86* 0.86* 0.68† 0.89* 0.86* 0.79† 0.89*Betaproteo. AUT −0.63* −0.60* −0.56* −0.44† −0.60* −0.45† −0.70** IleumControl −0.75† −0.82* −0.54 −0.61 −0.57 −0.39 −0.61 Betaproteo. AUT−0.56* −0.59* −0.64* −0.51† −0.61* −0.61* −0.85** Cecum Control −0.43−0.43 0.14 −0.00 0.14 0.14 −0.00 Spearman correlations are shown for theAUT-GI group alone (AUT) and the Control-GI group alone (Control). •= p< 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001, †= p < 0.1(trend) ^(a)= values obtained from bacteria-specific real-time PCR

To determine whether reductions in disaccharidase and transportertranscript levels reflected loss of or damage to intestinal epithelialcells, mRNA levels associated with a tissue-specific marker restrictedto these cells, villin (Khurana and George, 2008) were measured. Healvillin mRNA levels were not decreased in AUT-GI children (Mann-Whitney,p=0.307) (FIG. 16F). Normalization of SI, MGAM, LCT, SGLT1 and GLUT2 tovillin mRNA did not correct AUT-GI deficits in gene expression for thesetranscripts (FIGS. 22A-22E).

CDX2, a member of the caudal-related homeobox transcription factorfamily, regulates expression of SI, LCT, GLUT2, SGLT1 and villin (Suhand Traber, 1996; Troelsen et al., 1997; Uesaka et al., 2004;Balakrishnan et al., 2008; and Yamamichi et al., 2009). Real-time PCRexperiments demonstrated lower levels of CDX2 mRNA in some AUT-GIsubjects as compared with controls, but group differences were notsignificant (FIG. 16G: Mann-Whitney, p=0.192). Only 33.3% of AUT-GIpatients had CDX2 mRNA levels below the 25^(th) percentile of theControl-GI group (FIG. 23A). However, 86.7% of AUT-GI children had CDX2levels below the 50^(th) percentile of Control-GI children. Only oneAUT-GI child (patient 7) had CDX2 levels above the 75^(th) percentile ofControl-GI children. This child was the only subject who did not showsigns of deficiencies in any disaccharidases or transporters (Table 5A).In the AUT-GI group, expression of CDX2 was correlated with that of SI,MGAM, LCT, SGLT1, GLUT2, and villin (Bonferroni-adjusted Spearman rankorder correlations; Table 2). Among Control-GI subjects, the expressionof CDX2 was correlated only with that of MGAM, LCT, SGLT1, and villin(Table 2).

To determine whether deficient carbohydrate digestion and absorptioninfluenced the composition of intestinal microflora, ileal and cecalbiopsies from AUT-GI and Control-GI children were analyzed by bacterial16S rRNA gene pyrosquencing (See also Methods and FIGS. 23A-23D).Bacteroidetes and Firmicutes were the most prevalent taxa present in theileal and cecal tissues of AUT-GI children, with the exception of theileal samples of patients 2, 15, and 19 and cecal samples of patient 15,wherein levels of Proteobacteria exceeded those of Firmicutes and/orBacteroidetes (FIGS. 17A-B and FIGS. 24A-B). Other phyla identified atlower levels included Verrucomicrobia, Actinobacteria, Fusobacteria,Lentisphaerae, TM7, and Cyanobacteria, as well as unclassified bacterialsequences (FIGS. 17A-B and FIGS. 24A-24D). The abundance ofBacteroidetes was lower in AUT-GI ileal (FIG. 17C: Mann-Whitney,p=0.012) and cecal samples (FIG. 17D: Mann-Whitney, p=0.008) as comparedwith the abundance of Bacteroidetes in Control-GI samples. Real-time PCRusing Bacteroidete-specific primers confirmed decreases in Bacteroidetesin AUT-GI ilea (FIG. 17E: Mann-Whitney, p=0.003) and ceca (FIG. 17F:Mann-Whitney, p=0.022), with levels below the 25^(th) percentile ofControl-GI children in 100% of AUT-GI ilea and 86.7% of AUT-GI ceca(Table 5B). Family-level analysis of Bacteroidete diversity frompyrosequencing reads indicateed that losses among members of the familyBacteroidaceae in AUT-GI patient samples contributed substantially tooverall decreases in Bacteroidete levels in ilea (FIG. 17G) and ceca(FIG. 17H). OTU (Operational Taxonomic Unit) analysis of Bacteroidetesequences indicated that deficiencies in Bacteroidete sequences inAUT-GI subjects were attributable to cumulative losses of 12 predominantphylotypes of Bacteroidetes, rather than loss of any one specificphylotype (FIGS. 25A-25E and Methods).

Analysis of pyrosequencing reads revealed an increase inFirmicute/Bacteroidete ratios in AUT-GI ilea (FIG. 18A: Mann-Whitney,p=0.026) and ceca (FIG. 18B: Mann-Whitney, v0.032). An increase was alsoobserved at the order level for Clostridiales/Bacteroidales ratios inilea (FIG. 26A: Mann-Whitney, p=0.012) and ceca (FIG. 26B: Mann-Whitney,v0.032). Real-time PCR using Firmicute- and Bacteroidete-specificprimers confirmed these increases in Firmicute/Bacteroidete ratios inAUT-GI ilea (FIG. 26C: Mann-Whitney, v0.0006) and ceca (FIG. 26D:Mann-Whitney, p=0.022). Firmicute/Bacteroidete ratios were above the75^(th) percentile of Control-GI values in 100% of AUT-GI ilea and 60%of AUT-GI ceca (Table 5C). Order-level analysis of pyrosequencing readsindicated trends toward increased Clostridiales in AUT-GI ilea (FIG.27E: Mann-Whitney, p=0.072) and ceca (FIG. 27F: Mann-Whitney, p=0.098).Family-level analysis revealed that increased Clostridiales levels inAUT-GI patient samples were largely attributable to increases in membersof the families Lachnospiraceae and Ruminococcaceae (FIGS. 18C-18F).Cumulative levels of Lachnospiraceae and Ruminococcaceae above the75^(th) percentile of the corresponding levels in Control-GI sampleswere found in 60% of AUT-GI ileal and 53.3% of AUT-GI cecal samples(FIGS. 18E-18F and Table 5B). Genus-level analysis indicated thatmembers of the genus Faecalibacterium within the family Ruminococcaceaecontributed to the overall trend toward increased Clostridia levels(FIGS. 28A-B). Within Lachnospiraceae, members of the genusLachnopsiraceae Incertae Sedis, Unclassified Lachnospiraceae, and to alesser extent Bryantella (cecum only) contributed to the overall trendtoward increased Clostridia in ASD-GI patients (FIGS. 28A-B).

The cumulative level of Firmicutes and Proteobacteria was higher inAUT-GI group in both ileal (FIG. 18G: Mann-Whitney, p=0.015) and cecalsamples (FIG. 18H: Mann-Whitney, p=0.007) (FIGS. 18I-J); however,neither Firmicute nor Proteobacteria levels showed significantdifferences on their own (FIGS. 19A-19B and FIGS. 27A-27D). Levels ofBetaproteobacteria tended to be higher in the ilea of AUT-GI patients(FIG. 19C: Mann-Whitney, p=0.072); significantly higher levels ofBetaproteobacteria were found in AUT-GI ceca (FIG. 19D: Mann-Whitney,p=0.038). Levels of Betaproteobacteria were above the 75^(th) percentileof Control-GI children in 53.3% of AUT-GI ilea and 66.7% of AUT-GI ceca(Table 5B). Family-level analysis revealed that members of the familiesAlcaligenaceae and Incertae Sedis 5 (patient 2 only) contributedsubstantively to the observed increases in Beta-Proteobacteria in ilea(FIG. 19E) and ceca (FIG. 19F). Alcaligenaceae sequences were detectedin 46.7% of AUT-GI children and none of the Control-GI children. Overtlyelevated levels of Proteobacteria in AUT-GI ilea and ceca reflectedincreased Alpha- (families Methylo-bacteriaceae and UnclassifiedRhizobiales) and Betaproteobacteria (family Incertae Sedis 5) forpatient #2 and increased Gammaproteobacteria (family Enterobacteriaceae)for patients #8 and #15 (FIGS. 19E-19F). Levels of Alpha-, Delta-,Gamma-, and Epsilonproteobacteria were not significantly differentbetween AUT-GI and Control-GI samples.

The relationships between ileal and cecal microflora and levels ofdisaccharidases, transporters, villin, and CDX2 were assessed (Table 2).In the AUT-GI group, significant inverse Spearman correlations werefound for ileal Firmicutes vs. SI and MGAM; the ilealFirmicute/Bacteroidete ratio vs. SI, MGAM, LCT, SGLT1, GLUT2, and CDX2;and ileal and cecal Betaproteobacteria vs. SI, MGAM, LCT, GLUT2, andCDX2. In the Control-GI group significant inverse Spearman correlationswere found for cecal Bacteroidetes vs. SI, MGAM, SGLT1, and CDX2; aswell as ileal Betaproteobacteria vs. MGAM. Positive Spearmancorrelations were also found in the Control-GI group: cecal Firmicutesvs. SI, MGAM, SGLT1, GLUT2, and CDX2; and cecal Firmicute/Bacteroideteratio vs. SI, MGAM, SGLT1, GLUT2, and CDX2 (Table 2). These resultsindicate a complex dependence between carbohydrate metabolizing andtransporting genes and the composition of the intestinal microbiome (SeeFIG. 20A-20C).

Discussion

ASD are brain disorders defined using behavioral criteria; however, manyaffected individuals also have substantial GI morbidity. A previousreport on GI disturbances in ASD found low activities of at least onedisaccharidase or glucoamylase in duodenum in 58% of children examined(21 of 36) (Horvath et al., 1999). As described herein, 93.3% of AUT-GIchildren had decreased mRNA levels for at least one of the threedisaccharidases (SI, MGAM, or LCT). In addition, decreased levels ofmRNA were found for two important hexose transporters, SGLT1 and GLUT2.Transcripts for the enterocyte marker, villin, were not deficient inAUT-GI ilea; thus these deficiencies are unlikely to be due to a generalloss of enterocytes. However, defects in enterocyte maturational ormigration along the crypt-villus axis can compromise ranscriptionalregulation of ileal enzymes and transporters (Hodin et al., 1995). Theexpression of CDX2, a master transcriptional regulator in the intestine,was correlated with expression of disaccharidases and transporters inAUT-GI children. Therefore, CDX2 could play a role in the observedexpression deficits for these genes. Whatever the mechanism, reducedcapacity for digestion and transport of carbohydrates can have profoundeffects. Within the intestine malabsorbed monosaccharides can lead toosmotic diarrhea; non-absorbed sugars can also serve as substrates forintestinal microflora that produce fatty acids and gases (methane,hydrogen, and carbon dioxide), promoting additional GI symptoms such asbloating and flatulence. The deficiency of even a single gene in thisimportant pathway can result in severe GI disease, as occurs withGlucose-galactose malabsorption syndrome caused by SGLT1 deficiency,Fanconi-Bickel syndrome resulting from GLUT2 mutations,sucrase-isomaltase deficiency, and congenital lactase deficiency.Without being bound by theory, a potential link between neurologicaldysfunction and malabsorption in childhood autism has been indicated(Goodwin et al., 1971). Extra-intestinal manifestations of GI disease,including neurologic presentation, are described in patients withinflammatory bowel disease and celiac disease (Bushara 2005; Lossos etal., 1995; Gupta et al., 2005). An association between languageregression and GI symptoms has been reported in ASD, supporting a linkbetween GI disease and behavioral outcomes (Valicenti-McDermott et al.,2008). Outside the intestine, the major role of dietary carbohydrates isto serve as the primary source of cellular energy throughout the body.Following digestion, nearly all ingested carbohydrates are converted toglucose, which serves a central role in metabolism and cellularhomeostasis. The brain, of all organs, is quantitatively the mostenergy-demanding, accounting for 50% of total body glucose utilization(Owen et al., 1967). Abnormalities in glucose metabolism and homeostasishave been documented in ASD: recovery of blood glucose levels wasdelayed in ASD children following insulin-induced hypoglycemia (Maher etal., 1975). Brain glucose metabolism is decreased in ASD by positronemission tomography (Toal et al., 2005; Haznedar et al., 2000; Haznedaret al., 2006). Without being bound by theory, a reduced capacity todigest carbohydrates and absorb glucose due to deficient expression ofdisaccharidases and hexose transporters explains these previousobservations in ASD.

Changes in diet can influence composition of intestinal microflora;thus, without being bound by theory carbohydrate malabsorption can havesimilar effects in AUT-GI subjects. 16S rRNA pyrosequencing revealedmulticomponent dysbiosis in AUT-GI children including decreased levelsof Bacteroidetes, an increase in the Firmicute/Bacteroidete ratio,increased cumulative levels of Firmicutes and Proteobacteria, and anincrease in the class Betaproteobacteria. Bacteroidetes are implicatedin mediating maturational and functional processes in the intestine aswell as immune modulation. Monocolonization of mice with the prototypicgut symbiont, Bacteroides thetaiotaomicron, reverses the maturationaldefect in ileal epithelial glycan fucosylation that occurs in germ-freemice and regulates the expression of host genes, including SGLT-1 andLCT, that participate in key intestinal functions (i.e., nutrientabsorption, metabolism, epithelial barrier function, and intestinalmaturation) (Hooper et al., 2001).

A direct role for Bacteroidetes in carbohydrate metabolism is alsoevident. B. thetaiotaomicron encodes in its genome an expansive numberof genes dedicated to polysaccharide acquisition and processing,including 236 glycoside hydrolases and 15 polysaccharide lyases (Flintet al., 2008). Thus, deficient digestion and absorption of di- andmonosaccharides in the small intestine can alter the milieu of growthsubstrates in the ileum and cecum. As such, the growth advantages thatBacteroidetes enjoy in the healthy intestine as a result of theirexpansive capacity to thrive on polysaccharides can be compromised inAUT-GI children as bacterial species better suited for growth onundigested and unabsorbed carbohydrates flourish. Furthermore,polysaccharide A (PSA), a single molecule from another Bacteroidetemember, Bacteroides fragilis, protects germ-free mice from Helicobacterhepaticus- and chemically-induced colitis by correcting defects inT-cell development, suppressing production of IL-17 and TNF-alpha, andinducing IL-10 (Mazmanian et al., 2008). These reports highlight themultiple roles Bacteroidete members play in the maintenance ofintestinal homeostasis, including maturation of epithelium; regulationof intestinal gene expression, including carbohydrate metabolizing genesand transporters; metabolism of polysaccharides in the colon; anddevelopment of a competent immune system. Thus, deficient levels ofBacteroidetes in the muco-epithelium of AUT-GI children can directlycompromise carbohydrate metabolism and trigger inflammatory pathways.

Mice that are genetically obese (ob/ob) have 50% fewer Bacteroidetes. Alower abundance of Bacteroidetes is reported in stool samples from obeseindividuals (Ley et al., 2005; Ley et al., 2006). UsingBacteroidete-specific real-time PCR, dramatic decreases were found inthe ilea (˜50% lower abundance) as well as significantly lower levels inthe ceca (˜25% lower abundance) of AUT-GI compared to Control-GIchildren. In ob/ob mice, diet-induced obese mice, and in obese humans,the decrease in Bacteroidetes is accompanied by an increase inFirmicutes (Turnbaugh et al., 2008; Ley et al., 2005; Ley et al., 2006).The increased Firmicute/Bacteroidete ratio in obesity increases thecapacity to harvest energy from the diet (Turnbaugh et al., 2006). Asdiscussed herein, the trend toward increased Firmicutes and thesignificant decrease in Bacteriodetes led to a significant increase inthe Firmicute/Bacteroidete ratio in ilea and ceca of AUT-GI compared toControl-GI children. The trend toward increased Firmicutes was largelyattributable to Clostridia members; based on pyrosequencing result,members of Ruminococcaceae and Lachnospiraceae were the majorcontributors.

Several members of Ruminococcaceae and Lachnospiraceae are knownbutyrate producers and can thus influence short-chain fatty acid (SCFA)levels (Louis et al., 2010). SCFA influence colonic pH and Bacteroidessp. are relatively sensitive to acidic pH (Duncan et al., 2009). Threereports indicated differences in Clostridia species in stool samplesfrom ASD-GI as compared to control children, including greater abundanceof Clostridium clusters I, II, XI and C. bolteae (Finegold et al, 2002;Song et al., 2004; Parracho et al., 2005). Although only a trend wasobserved for increased Firmicutes in AUT-GI children, the cumulativelevels of Firmicutes and Proteobacteria were significantly higher. ThreeAUT-GI patients had extremely high levels of Alpha- and Beta-, orGammaproteobacteria. In addition, the AUT-GI group had elevated levelsof Betaproteobacteria compared to the Control-GI group, reflecting thepresence of Alcaligenaceae members in the ilea and ceca of 46.7% ofAUT-GI children. Alcaligenaceae sequences were not detected in tissuesfrom Control-GI children.

Conclusions:

Metabolic interactions between intestinal symbionts and the human hostare only beginning to be understood. Increasing evidence shows thatgastrointestinal disease and dysbiosis exert system-wide effects onnormal host physiology. As discussed herein, GI disease in autism has amolecular profile distinct from GI disease in normally-developingchildren. AUT-GI children have deficiencies in disaccharidase and hexosetransporter gene expression that likely promote malabsorption andmulticomponent, compositional dysbiosis. Although the extra-intestinaleffects these changes can elicit remain speculative, the identificationof specific molecular and microbial signatures that definegastrointestinal pathophysiology in AUT-GI children sets the stage forfurther research aimed at defining the epidemiology, diagnosis andinformed treatment of GI symptoms in autism.

Materials and Methods:

Patient samples. Patient biopsies were collected as part of a study toassess the frequency of measles virus transcripts in ilea and ceca ofchildren with autistic disorder and gastrointestinal complaints (AUT-GI,n=15) and children with gastrointestinal complaints without braindisorder (Control-GI, n=7). This cohort has been previously described indetail (Hornig et al., 2008). The present study restricted to male,Caucasian children from the original cohort between 3 and 5 years of ageto control for confounding effects of gender, race and age on intestinalgene expression and bacterial microbiota. The age at biopsy was similarfor AUT-GI and Control-GI subjects (median, in years [interquartilerange, IQR]: AUT-GI, 4.5 (1.2); Control-GI, 3.98 (0.9); Mann-Whitney,p=0.504] (See Table 3).

TABLE 3 Patient information Table. Age LCT Patient # Group (yrs.)(13910:22018) 215 1 ASD 4.35 C/T:G/A 478 2 ASD 5.94 T/T:A/A 513 3 ASD4.66 T/T:A/A 530 4 ASD 5.46 C/T:G/A 554 5 ASD 4.01 T/T:A/A 562 6 ASD3.80 C/T:G/A 566 7 ASD 3.49 T/T:A/A 581 8 ASD 4.29 T/T:A/A 589 9 ASD5.62 C/C:G/G* 648 10 ASD 4.71 C/T:G/A 678 11 ASD 5.28 T/T:A/A 686 12 ASD5.03 C/T:G/A 688 13 ASD 4.00 C/C:G/G* 733 14 ASD 4.53 T/T:A/A 800 15 ASD3.51 C/C:G/G* 667 16 Control 3.98 T/T:A/A 755 17 Control 5.06 T/T:A/A760 18 Control 3.89 C/T:G/A 796 19 Control 5.48 C/T:G/A 797 20 Control3.98 C/T:G/A 814 21 Control 3.95 C/C:G/G* 842 22 Control 4.12 T/T:A/A

RNA and DNA extraction. RNA and DNA were extracted sequentially fromindividual ileal and cecal biopsies (total of 176 biopsies: 88 ileal and88 cecal biopsies; 4 biopsies per patient per region; 15 AUT-GI patientsand 7 Control-GI patients) in TRIzol using standard protocols. RNA andDNA concentrations and integrity were determined using a NanodropND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, Del.) andBioanalyzer (Agilent Technologies, Foster City, Calif.) and stored at−80° C.

Quantitative Real-Time PCR of human mRNA. Intron/exon spanning,gene-specific PCR primers and probes for sucrase isomaltase, maltaseglucoamylase, lactase, SGLT1, GLUT2, Villin, and CDX2, with GAPDH andBeta-actin as dual housekeeping gene controls were designed forreal-time PCR using Primer Express 1.0 software (Applied Biosystems,Foster City, Calif.). Taqman probes were labeled with the reporter FAM(6-carboxyfluorescein) and the quencher BBQ (Blackberry) (TIB MolBiol).PCR standards for determining copy numbers of target transcripts weregenerated from amplicons cloned into the vector pGEM-T easy (PromegaCorporation, Madison, Wis.). Linearized plasmids were quantitated by UVspectroscopy and 10-fold serial dilutions (ranging from 5×10⁵ to 5×10⁰copies) were created in water containing yeast tRNA (1 ng/μl). UnpooledRNA from individual ileal biopsies were used for real time PCR assays;each individual biopsy was assayed in duplicate. cDNA was synthesizedusing Taqman reverse transcription reagents (Applied Biosystems) from 2μg unpooled RNA per 100 μl reaction. Each 25-μl amplification reactioncontained 10 μl template cDNA, 12.5 μl Taqman Universal PCR Master Mix(Applied Biosystems), 300 nM gene-specific primers and 200 nMgene-specific probe (Table 2). The thermal cycling profile using a ABIStepOnePlus Real-time PCR System (Applied Biosystems) consisted of:Stage 1, one cycle at 50° C. for 2 min; Stage 2, 1 cycle at 95° C. for10 min; Stage 3, 45 cycles at 95° C. for 15 s and 60° C. for 1 min (1min 30 s for LCT). GAPDH and B-actin mRNA were amplified in duplicatereactions by real-time PCR from the same reverse transcription reactionas was performed for the gene of interest. The mean concentration ofGAPDH or Beta-actin in each sample was used to control for integrity ofinput RNA and to normalize values of target gene expression to those ofthe housekeeping gene expression. The final results shown were expressedas the mean copy number from replicate biopsies per patient, relative tovalues obtained for GAPDH mRNA. Beta-actin normalization gave similarresults to GAPDH normalization for all assays. Due to insufficient orpoor quality RNA, only 3 of the 4 biopsies were included for 3 patients(Patient #s 4, 7, 10) and only 2 of the 4 biopsies were included for 1patient (Patient #2). Thus, 83 of the original 88 ileal biopsies wereused in real-time PCR experiments.

Lactase genotyping. Genomic DNA from AUT-GI (n=15) and Control-GI (n=7)patients was subjected to previously-described PCR-restriction fragmentlength polymorphism (PCR-RFLP) analysis for the C/T-13910 and G/A-22018polymorphisms associated with Adult-type Hypolactasia with minormodifications (Buning et al., 2003). Genotyping primers for C/T-13910and G/A-22018 polymorphisms are as follows: C/T-13910 For(5′-GGATGCACTGC TGTGATGAG-3′[SEQ ID NO: 20]), C/T-13910Rev(5′-CCCACTGACCTATCCTCGTG-3′ [SEQ ID NO: 21]), G/A-22018 For(5′-AACAGGCACGTGGAGGAGTT-3′ [SEQ ID NO: 22]), and G/A-22018Rev(5′-CCCACCTCAGCCTCTTGAGT-3′[SEQ ID NO: 23]). Each 50-μl amplificationreaction contained 500 ng genomic DNA, 400 nM forward and reverseprimers, and 25 μl High Fidelity PCR master mix. Thermal cyclingconsisted of 1 cycle at 94° C. for 4 min followed by 40 cycles at 94° C.for 1 min, 60° C. for 1 min, and 72° C. for 1 min. PCR reactions forC/T-13910 were directly digested with the restriction enzyme BsmFI at65° C. for 5 hrs. PCR reactions for G/A-22018 were resolved on 1%agarose gels followed by gel extraction of the prominent 448 bpamplicon. Gel extracted G/A-22018 amplicons were then digested with therestriction enzyme HhaI at 37° C. for 5 hrs. Restriction digests ofC/T-13910 and G/A-22018 were resolved on 1.5% ethidium-stained agarosegels for genotyping analysis. BsmFI digestion of the C/T-13910 ampliconsgenerates two fragments (351 bp and 97 bp) for the hypolactasia genotype(C/C), four fragments (351 bp, 253 bp, 98 bp, and 97 bp) for theheterozygous genotype (C/T), and three fragments (253 bp, 98 bp, and 97bp) for the normal homozygous allele (T/T). HhaI digestion of theG/A-22018 amplicons generates two fragments (284 bp and 184 bp) for thehypolactasia genotype (G/G), three fragments (448 bp, 284 bp, and 184bp) for the heterozygous genotype (G/A), and a single fragment (448 bp)for the normal homozygous allele (A/A).

PCR amplification of bacterial 16S rRNA gene and barcoded 454pyrosequencing of intestinal microbiota. For DNA samples from 88 ilealbiopsies (4 biopsies per patient; 15 AUT-GI patients, 7 Control-GIpatients) and 88 cecal biopsies from the same patients, PCR was carriedout using bacterial 16S rRNA gene-specific (V2-region), barcoded primersas previously described (Hamady et al., 2008). Composite primers were asfollows: (For) 5′-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3′[SEQ IDNO: 24], (Rev) 5′-GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3′[SEQ ID NO: 25]. Underlined sequences in the Forward and Reverse primersrepresent the 454 Life Sciences@ primer B and primer A, respectively.Bold sequences in the forward and reverse primers represent thebroadly-conserved bacterial primer 27F and 338R, respectively. NNNNNNNNrepresents the eight-base barcode, which was unique for each patient.PCR reactions consisted of 8 μl 2.5×5 PRIME HotMaster Mix (5 PRIME Inc.,Gaithersburg, Md.), 6 μl of 4 μM forward and reverse primer mix, and 200ng DNA in a 20-μl reaction volume. Thermal cycling consisted of onecycle at 95° C. for 2 min; and 30 cycles at 95° C. for 20 seconds, 52°C. for 20 seconds, and 65° C. for 1 min. Each of 4 biopsies per patientwas amplified in triplicate, with a single, distinct barcode applied perpatient. Ileal and cecal biopsies were assayed separately. Triplicatereactions of individual biopsies were combined, and PCR products werepurified using Ampure magnetic purification beads (Beckman CoulterGenomics, Danvers, Mass.) and quantified with the Quanti-iT PicoGreendsDNA Assay Kit (Invitrogen, Carlsbad, Calif.) and Nanodrop ND-1000Spectrophotometer (Nanodrop Technologies, Wilmington, Del.). Equimolarratios were combined to create two master DNA pools, one for ileum andone for cecum, with a final concentration of 25 ng/μl. Master pools weresent for unidirectional pyrosequencing with primer A at 454 LifeSciences (Branford, Conn.) on a GS FLX sequencer.

Real-time PCR of Bacteroidete and Firmicute 16S rRNA genes. Primersequences used for real-time PCR are listed in Table 4.

TABLE 4 Real-time PCR primers and probes used for gene expressionand bacterial quantitative analysis. SEQ Amplicon size Name ID NO.Primers and Probe (bp) SI 26 For: 5′-TCTTCATGAGTTTTATGAGGATACGAAC-3′ 15027 Rev: 5′-TTTGCACCAGATTCATAATCATACC-3′ 28 Probe:5′-CAGATACTGTGAGTGCCTACATCCCTGATGCTATT-3′ MGAM 29 For:5′-TACCTTGATGCATAAGGCCCA-3′ 150 30 Rev: 5′-GGCATTACGCTCCAGGACA-3′ 31Probe: 5′-CGTCACTGTTGTGCGGCCTCTGC-3′ LCT 32 For:5′-CAGGAATCAAGAGCGTCACAACT-3′ 180 33 Rev: 5′-AAATCGACCGTGTCCTGGG-3′ 34Probe: 5′-TCCTGCTAGAACCACCCATATCTGCGCT-3′ SGLT1 35 For:5′-GCTCATGCCCAATGGACTG-3′ 125 36 Rev: 5′-CGGACCTTGGCGTAGATGTC-3′ 37Probe: 5′-ACAGCGCCAGCACCCTCTTCACC-3′ Glut2 38 For:5′-AGTTAGATGAGGAAGTCAAAGCAA-3′ 164 39 Rev: 5′-TAGGCTGTCGGTAGCTGG-3′ 40Probe: 5′-ACAAAGCTTGAAAAGACTCAGAGGATATGATGATGTC-3′ Villin 41 For:5′-CATGCGCTGAACTTCATCAAA-3′ 120 42 Rev: 5′-GGTTGGACGCTGTCCACTTC-3′ 43Probe: 5′-CGGCCGTCTTTCAGCAGCTCTTCC-3′ CDX2 44 For:5′-GGCAGCCAAGTGAAAACCAG-3′ 112 45 Rev: 5′-TCCGGATGGTGATGTAGCG-3′ 46Probe: 5′-ACCACCAGCGGCTGGAGCTGG-3′ β-Actin 47 For:5′-AGCCTCGCCTTTGCCGA-3′ 175 48 Rev: 5′-CTGGTGCCTGGGGCG-3′ 49 Probe:5′-CCGCCGCCCGTCCACACCCGCC GAPDH 50 For: 5′-CCTGTTCGACAGTCAGCCG-3′ 100 51Rev: 5′-CGACCAAATCCGTTGACTCC-3′ 52 Probe: 5′-CGTCGCCAGCCAGAGCCACA-3′Bacteroidetes 53 For: 5′-AACGCTAGCTACAGGCTT-3′ ~293(Frank et al.) 54Rev: 5′-CCAATGTGGGGGACCTTC-3′ Firmicutes 55 For:5′-GGAGYATGTGGTTTAATTCGAAGCA-3′ ~126(Guo et al.) 56 Rev:5′-AGCTGACGACAACCATGCAC-3′ Total Bacteria 57 For:5′-GTGCCAGCMGCCGCGGTAA-3′ ~295(Frank et al.) 58 Rev:5′-GACTACCAGGGTATCTAAT-3′

PCR standards for determining copy numbers of bacterial 16S rDNA wereprepared from representative amplicons of the partial 16S rRNA genes ofBacteroidetes and Firmicutes and total Bacteria cloned into the vectorPGEM-T easy (Promega). A representative amplicon with high homology toBacteroides Vulgatus (Accession #: NC_(—)009614) was used withBacteroidete-specific primers. A representative amplicon with highhomology to Faecalibacterium prausnitzii (Accession #: NZ_ABED02000023)was used with Firmicute-specific primers. A representative amplicon withhigh homology to Bacteroides intestinalis (Accession #: NZ_ABJL02000007)16S rRNA gene was used with total Bacteria primers. Cloned sequenceswere classified using the RDP Seqmatch tool and confirmed by theMicrobes BLAST database. Plasmids were linearized with the SphIrestriction enzyme and ten-fold serial dilutions of plasmid standardswere created ranging from 5×10⁷ to 5×10⁰ copies for Bacteroidetes,Firmicutes and total Bacteria. Amplification and detection of DNA byreal-time PCR were performed with the ABI StepOnePlus Real-time PCRSystem (Applied Biosystems). Cycling parameters for Bacteroidetes andtotal Bacteria were as previously described (Frank et al., 2007), aswere cycling parameters for Firmicutes (Guo et al., 2008). Each 25-μlamplification reaction mixture contained 50 ng DNA, 12.5 μl SYBR GreenMaster Mix (Applied Biosystems), and 300 nM bacteria-specific(Bacteroidete, Firmicute or total Bacteria) primers. DNA from each of 88ileal biopsies (4 biopsies per patient) and 88 cecal biopsies (4biopsies per patient) was assayed in duplicate. The final results wereexpressed as the mean number of Bacteroidete or Firmicute 16S rRNA genecopies normalized to 16S rRNA gene copies obtained using total Bacterialprimers. Eight water/reagent controls were included for allamplifications. The average copy number for water/reagent controls(background) was subtracted from each ileal and cecal amplificationprior to normalization. For the Bacteroidete assay all water controlscontained undetectable levels of amplification. For the Firmicute assayaverage amplification signal from water samples were minimal,12.03+/−15.0 copies.

Bioinformatic analysis of pyrosequencing reads. Pyrosequencing readsranging from 235 to 300 base pairs in length (encompassing all sequenceswithin the major peak obtained from pyrosequencing) were filtered foranalysis. Low-quality sequences—i.e., those with average quality scoresbelow 25—were removed based on previously described criteria (Huse etal., 2007; Hamady et al., 2008). Additionally, reads with any ambiguouscharacters were omitted from analysis. Sequences were then binnedaccording to barcode, followed by removal of primer and barcodesequences. Taxonomic classifications of bacterial 16S rRNA sequenceswere obtained using the RDP Classifier with a minimum 80% bootstrapconfidence estimate. To normalize data for differences in totalsequences obtained per patient, phylotype abundance was expressed as apercentage of total bacterial sequence reads per patient at alltaxonomic levels.

Statistical analysis. Data were not normally distributed, based onKolmogorov-Smirnov test and evaluation of skewness and kurtosis; thus,the non-parametric Mann-Whitney U test was performed using StatView(Windows version 5.0.1; SAS Institute, Cary, N.C.). The comparativeresults of gene expression and bacteria levels were visualized asbox-and-whisker plots showing: the median and the interquartile(midspread) range (boxes containing 50% of all values), the whiskers(representing the 25^(th) and 75^(th) percentiles) and the extreme datapoints (open circles). Associations between different variables wereassessed by Spearman rank correlation test. Chi-squared test was used toevaluate between-group genotypes for adult-type hypolactasia.Kruskal-Wallis one-way analysis of variance was employed to assesssignificance of LCT mRNA expression levels split by genotype and group.Significance was accepted at p<0.05.

Genetically determined lactase non-persistence is not responsible fordeficient lactase mRNA in AUT-GI. Although it is beyond the scope ofthis study to evaluate all possible mutations in carbohydrate genes thatcan affect expression, deficient LCT mRNA is not a result of the commonadult-type hypolactasia genotype. LCT mRNA levels can be affected by twosingle nucleotide polymorphisms that determine adult-type hypolactasia;therefore, we genotyped these children using PCR-RFLP analysis (FIG.21A). The homozygous, hypolactasia variant alleles were found in 20% (3out of 15) of AUT-GI children and 14.3% (1 out of 7) of Control-GIchildren (chi-squared test, p=0.896) (FIG. 21B). LCT mRNA expression wassignificantly lower in individuals with the homozygous hypolactasiagenotype compared to all other genotypes (FIG. 21C: Mann-Whitney,p=0.033). Comparison of LCT mRNA expression across genotype and groupfailed to reach significance (FIG. 21D: Kruskal-Wallis, p=0.097).Comparison of mRNA expression in subjects carrying at least one copy ofthe normal allele confirmed a significant decrease in LCT mRNA in AUT-GIrelative to Control-GI subjects, independent of the individuals with thehomozygous hypolactasia genotype (FIG.21E: Mann-Whitney, p=0.025). Insummary, although the data support the notion that LCT genotype affectsgene expression, deficient LCT mRNA in AUT-GI was not attributable todisproportionate hypolactasia genotypes between the AUT-GI andControl-GI groups.

Barcoded 16S rRNA gene pyrosequencing. A total of 525,519 sequencingreads (representing 85% of the initial number of sequencing reads)remained after filtering based on read length, removing low-qualitysequences and combining duplicate pyrosequencing runs (271,043 reads forilea; 254,476 reads for ceca). Binning of sequences by barcode revealedsimilar numbers of 16S rRNA gene sequence reads per patient (average #sequences per patient+/−STD for ilea=12,320+/−1220; average # sequencesper patient+/−STD for ceca=11,567+/−1589). There was not a significantdifference between the AUT-GI and Control-GI groups in terms of thenumber of reads per patient. In order to assess whether sufficientsampling was achieved in the total pyrosequencing data set for allAUT-GI and Control-GI subjects, OTUs (Operational Taxonomic Units) weredefined at a threshold of 97% identity, split by data for ileum andcecum, and rarefaction analysis was carried out (FIGS. 23A-23B).Rarefaction curves showed a tendency toward reaching plateau for allsubjects; however failure to reach plateau means that additionalsampling would be required to achieve complete coverage of all OTUspresent in ileal and cecal biopsies. Investigation of diversity inAUT-GI and Control-GI patients was carried out using the ShannonDiversity Index calculated from OTU data for each subject. Rarefactionanalysis revealed that all Shannon Diversity estimates had reachedstable values (FIGS. 23C-23D). While Shannon Diversity estimates variedwidely between individuals, there was not an apparent overall difference(loss or gain of diversity) between the AUT-GI and Control-GI groups inileal (FIG. 23C) or cecal (FIG. 23D) biopsies.

OTU Analysis of Bacteroidetes. In order to determine whether thedecreased abundance of Bacteroidete members was attributable to the lossof specific Bacteroidete phylotypes, the distribution of BacteroideteOTUs (defined using a threshold of 97% identity or greater, 3% distance)was investigated. The number of Bacteroidete OTUs per patient rangedfrom 23 to 102 for ileal samples and 10 to 130 for cecal samples.Interestingly, no single OTU was significantly over or underrepresentedbetween AUT-GI and Control-GI children and many OTUs contained singlesequences. Thus, it was determined whether, the decrease in OTUs couldbe attributed to overall losses of the most prevalent Bacteroidetephylotypes. In both ileal and cecal samples, 12 OTUs accounted for themajority of Bacteroidete sequences (FIGS. 25A-25B). The cumulativelevels of these 12 OTUs were significantly lower in AUT-GI compared toControl-GI children in both the ileum (FIG. 25C: Mann-Whitney, p=0.008)and cecum (FIG. 25D: Mann-Whitney, p=0.008). Representative sequencesfrom each of these 12 OTUs were classified using Green Genes Blast(greengenes.lbl.gov) and microbial blast alignment (NCBI) (FIG. 25E).The majority of sequences were members of the family Bacteroidaceae(OTUs 3, 5, 6, 7, and 19), except in the case of patient 20, wherePrevotellaceae were the dominant phylotype. These results indicate thatthe loss of Bacteroidetes in AUT-GI children is primarily attributableto overall decreases in the dominant phylotypes of Bacteroidetes.

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Example 2 Intestinal Inflammation, Impaired Carbohydrate Metabolism andTransport, and Microbial Dysbiosis In Autism

The objective of this study was to survey host gene expression andmicroflora in intestinal biopsies from children with autistic disorderand gastrointestinal complaints (AUT-GI) vs children withgastrointestinal complaints alone (Control-GI).

This example herein describes a rapid and specific PCR-based assay fordiagnostic detection of Sutterella species in biological samples. It isa PCR-based detection scheme utilizing new genomic 16S rRNA sequences toallow rapid, sensitive, and specific species identification from gutsamples.

Overview

Methods. Transcription profiling was pursued by cDNA microarray usingRNA extracted from ileal biopsies (4 per patient) of 15 male AUT-GI and7 age-matched, male Control-GI patients. Pathway analysis was performedusing Ingenuity Pathway Analysis and GO Ontology. Changes in geneexpression were confirmed by quantitative real-time PCR. Intestinalmicrobiota were investigated in ileal and cecal biopsies from AUT-GI andControl-GI children using amplicon-based, bar-coded pyrosequencing ofthe V2 region of bacterial 16S rDNA. Taxonomic classification of 525,519bacterial sequences was accomplished using the Ribosomal DatabaseProject classifier tool. Differences in microbiota between the twogroups were further evaluated and confirmed using Bacteroidete-,Firmicute-, and Sutterella-specific real-time PCR.

Results. Microarray and pathway analysis revealed significant changes ingenes involved in carbohydrate metabolism and transport and inflammationin ileal biopsies from AUT-GI as compared to Control-GI subjects.Real-time PCR confirmed significant decreases in the AUT-GI group in theprimary brush border disaccharidases, sucrase isomaltase (p=0.0013),maltase glucoamylase (p=0.0027), and lactase (p=0.0316) as well as intwo enterocyte hexose transporters, sodium glucose co-transporter 1(p=0.0082) and glucose transporter 2 (p=0.0101). In contrast, increaseswere confirmed for inflammation-related genes in AUT-GI subjects:complement component 1, q subcomponent, A chain (p=0.0022), resistin(p=0.0316), CD163 (p=0.0150), tumor necrosis factor-like weak inducer ofapoptosis (p=0.015), and interleukin 17F (p=0.0220). No significantgroup differences were observed for the enterocyte-specific marker,villin. In conjunction with changes in intestinal gene expression,bacterial content differed between the AUT-GI and Control-GI groups:pyrosequencing and real-time PCR revealed lower levels of Bacteroidetes(ileum: 50% reduction, p=0.0027; cecum: 25% reduction, p=0.0220, andhigher Firmicute/Bacteroidete ratios in AUT-GI children (ileum:p=0.0006; cecum: p=0.0220). High levels of Sutterella species were foundin 47% of AUT-GI biopsies (7/15), whereas Sutterella was not detected inany Control-GI biopsies (0/7; ileum: p=0.0220; cecum: p=0.0368).

Conclusions. A syndrome in autistic children is described whereingastrointestinal dysfunction is associated with altered gene expressionreflecting intestinal inflammation, impaired carbohydrate metabolism andtransport, and dysbiosis. These findings provide insights intopathogenesis and allow for new strategies for therapeutic intervention.

In this study, high levels of Sutterella sp. were found in ileal andcecal biopsies from children with autism spectrum disorders (ASD) andgastrointestinal disease, while Sutterella sp. were undetectable incontrol children with gastrointestinal disease. Little is known aboutthe epidemiology and pathogenesis of Sutterella sp. and their role ininfectious diseases of humans and animals. Current methods for detectingSutterella sp. are costly, labor intensive, and non-specific requiringisolation and anaerobic culture of the bacteria or generation,screening, sequencing, and sequence analysis of hundreds to thousands ofbacterial 16S rRNA gene sequences from bacterial libraries orpyrosequencing analysis of hundreds of thousands of sequences. Thesemethods can be costly, lack specificity, ease of execution, and are notstrictly quantitative.

A rapid and specific PCR-based assay is described for the diagnosticidentification, quantification, and phylogenetic analysis of Sutterellasp. in biological samples based on the variable sequence (V6-V8 region)of the 16S rRNA gene of Sutterella sp.

Study Background

An association between autistic spectrum disorder (ASD) andgastrointestinal (GI) immunopathology is supported by reports of ahigher incidence of GI complaints, ileo-colonic lymphoid nodularhyperplasia, and enterocolitis in children with autism. In this study,intestinal bacteria were assessed in ileal (4 biopsies per patient) andcecal (4 biopsies per patient) biopsies from male ASD children (aged 3-5years) with gastrointestinal symptoms (ASD-GI; n=15) and normallydeveloping age-matched, male controls with gastrointestinal symptoms(Control-GI; n=7) by 454 pyrosequencing of the V2 region of thebacterial 16S rRNA gene. Taxonomic classification of 525,519 bacterialsequences was performed using the Ribosomal Database Project classifiertool. Genus-level analysis of pyrosequencing reads revealed asignificant increase in Sutterella sp. The average confidence estimateof all genus-level Sutterella sequences identified using the RDPClassifier was high (99.1%) with the majority of sequences at 100%confidence.

Comparison of ASD-GI and Control-GI patients revealed significantincreases in Sutterella sp. In the ileum (FIG. 8A: Mann-Whitney U,p=0.022) and cecum (FIG. 8B: Mann-Whitney U, v0.0368). Sutterella sp.sequences were completely absent from all Control-GI samples (% of totalbacteria=0). Individual analysis of ASD-GI patients revealed that 7 outof 15 ASD-GI patients (46.7%) had high levels of Sutterella sp.sequences in both the ileum and cecum (FIG. 8C and FIG. 8D). By patient,ileal Sutterella sp. sequence abundance ranged from 1.7 to 6.7% of totalbacterial reads (FIG. 8C). Similarly, in the Cecum Sutterella sp.sequence abundance ranged from 1.9 to 7.0% of total bacterial reads forthe same patients (FIG. 8D). Sutterella sp. Sequences represented themajority of sequences present in the class Beta-proteobacteria in theseselect ASD-GI patients. In the Ileum of these ASD-GI patients,Sutterella sp. sequences accounted for 75.6% to 97.8% of allBeta-proteobacteria sequences (FIG. 8E). In the cecum, Sutterella sp.sequences accounted for 92.7% to 98.2% of all Beta-proteobacteriasequences (FIG. 8F). The results of this costly, time consuming,non-specific pyrosequencing analysis prompted the design of a Sutterellasp.-specific PCR assay to confirm, quantitate, and determine taxonomy ofSutterella sp. in the same samples analyzed by pyrosequencing.

Methods

Primer and Probe Design: Sutterella sp.-specific 16S rRNA gene PCRprimers and probe were designed against the 16S sequence for Sutterellawadsworthensis (Genbank Accession #L37785) and Sutterella clone LW53(Genbank Accession #AY976224) using Primer Express 1.0 software (AppliedBiosystems, Foster City, Calif.). Genus specificity of candidate primerswas evaluated using the RDP Probe Match tool. While several potentialprimer pairs were identified, only one pair showed high specificity forSutterella sp. In PCR assays. These primers are designated here asSuttFor and SuttRev (Sequences of primers and probe are shown in Table1).

TABLE 1 Sutterella sp.-specific primers and probes for classical andreal-time PCR assays and pan-bacterial primers used for normalization.SEQ Amplicon size ID NO. Primers and Probe (bp) 11 SuttFor:5′-CGCGAAAAACCTTACCTAGCC-3′ ~260 12 SuttRev: 5′-GACGTGTGAGGCCCTAGCC-3′13 SuttProbe1: 5′-CACAGGTGCTGCATGGCTGTCGT-3′ 14 SuttProbe2:5′-CCG CAAGGGAATCTGGACACAGGT-3′ 15 515For: 5′-GTGCCAGCMGCCGCGGTAA-3′~295(Frank et al.) 16 805Rev: 5′-GACTACCAGGGTATCTAAT-3′

Evaluation of good quality sequences that were >1200 bases in the RDPdatabase revealed a total of 248 Sutterella sequences at the time ofanalysis. SuttFor and SuttRev_primers showed high exclusivity for thegenus Sutterella. Approximately 90% of RDP matches for SuttFor were inthe genus Sutterella and 100% of matches for the reverse primer wereSutterella sequences. The SuttFor primer sequence matched exactly withapproximately 91% (225/248 Sutterella sequences) of all Sutterellasequences, while the SuttRev primer matched exactly with approximately81% (200/248 Sutterella sequences) of all Sutterella sequences. TheSuttProbel (SEQ ID NO: 13) used for real-time PCR had low exclusivitybut high coverage of Sutterella sequences (100%). An additional probe(SEQ ID NO: 143) with high exclusivity, but low coverage of Sutterellasequences (58.8%) was also designed and can be used when sequenceinformation is available for Sutterella sp. in biological samples.

Classical PCR. The SuttFor and SuttRev primers amplify a 260 bp regionbetween variable regions 6, 7 and 8 (V6-V8) of the 16S rRNA ofSutterella. Classical PCR for detection of Sutterella was carried out in25 ul reactions consisting of 25 ng genomic DNA, 300 nm each SuttFor andSuttRev primers, 2 ul dNTP mix (10 mM; Applied Biosystems), 2.5 ul of10× PCR Buffer (Qiagen), 5 U of HotStarTaq DNA polymerase (Qiagen), and5 ul Q-solution (Qiagen). Cycling parameters consisted of an initialdenaturation step at 950 C for 15 min, followed by 30 cycles of 940 Cfor 1 min, 600 C for 1 min, and 720 C for 1 min and a final extension at720 C for 5 min. Amplified products were run on a 1.5% agarose gel,extracted from the gel and either sent for direct PCR product sequencingusing SuttFor and SuttRev primers or cloned into PGEM-T easy cloningvector for construction of bacterial libraries followed by sequencingusing vector primers. Specificity of the assay was confirmed throughdirect sequence analysis of PCR products and clone sequences using theRDP Seqmatch and Classifier tools. All PCR products and clones wereclassified as Sutterella by RDP. In order to test linearity andsensitivity of the assay, the Sutterella clone used for real-time PCRstandards was tested by classical PCR using the same conditions as allintestinal DNA. Ten fold dilutions of the_(—) Sutterella clone rangingfrom 5×105 to 5×100 were amplified by classical PCR alone as well asspiked into ileal DNA from a Sutterella negative patient. Both in thepresence and absence of background ileal DNA, the Classical PCR waslinear in the range of 5×105 to 5×102 copies and had an end-pointdetection limit of 5×101 copies (FIG. 9).

Quantitative Real-time PCR. PCR standards for determining copy numbersof bacterial 16S rDNA were prepared from representative clones of thepartial 16S rDNA of Sutterella obtained using the Classical PCR assay.Cloned sequences were classified using the RDP Seqmatch tool andconfirmed by the Microbes BLAST database. Plasmids were linearized withthe SphI restriction enzyme and ten fold serial dilutions of plasmidstandards were created ranging from 500,000 to 5 copies for Sutterella(FIG. 10A and FIG. 10B). Amplification and detection of DNA by real-timePCR were performed with the ABI StepOnePlus Real-time PCR System(Applied Biosystems). For Sutterella sp.-specific real-time PCR, each 25ul reaction contained 50 ng DNA, 12.5 ul Taqman universal master mix(ABI), 300 nm each of SuttFor and SuttRev primers, and 200 nm SuttProbel(Reporter=FAM, Quencher=BBQ). The standard curve had sensitivity down to5 copies ofplasmid, with a slope of −3.08, y-intercept of 41.787, andwith an R2 value of 0.996 (FIG. 10A and FIG. 10B). DNA from each of 88ileal biopsies and 88 cecal biopsies was assayed in duplicate. The finalresults were expressed as the mean number of copies normalized to 16SrRNA copies obtained using Pan-bacterial primers (Table 1: primers515For and 805Rev) in a SYBR Green Real-time PCR assay (see Ref. 6 formore information). While normalization to total bacteria is notnecessary, we have implemented its use in this study to control forvariation in input DNA. Eight water/reagent controls were included forall amplifications. The average copy number for water controls(background) was subtracted from each ileal and cecal amplificationprior to normalization. Where background copy number values exceededamplification values in ileal and cecal samples, copy number was set toa value of 0. Average amplification signal from water samples with theSutterella assay were very low (125.8+/−40 copies) compared toamplification in Sutterella positive samples (all ranging between 50,000and 1,000,000 copies). Average copy numbers for all ileum and cecumSutterella-negative amplifications was 26.6+/−21.0 copies (all werelower than the background controls).

Taxonomic Classification of Sutterella sp. Sequence alignments usingsequences obtained by direct sequencing of Sutterella sp. from theclassical PCR assay and phylogenetic analyses were conducted using MEGA4software. Primer sequences were trimmed from the sequences obtained bydirect sequencing of amplicons. Classification was confirmed using theRDP classifier and seqmatch tools. Sutterella sequences obtained fromileal and cecal biopsies were aligned with sequences from the 11 knownisolates of Sutterella sp. found in the RDP database. Sequences fromknown Sutterella sp. Isolates were trimmed to the length of thesequences obtained from ileal and cecal biopsies. Phylogenetic treeswere constructed according to the neighbour-joining method, rooted tothe outgroup Burkholderia pseudomallei, and the stability of thegroupings was estimated by bootstrap analysis (1000 replications) usingMEGA4.

Results

Implementation of Sutterella sp.-specific Classical PCR for Detection.Classical PCR analysis of Sutterella sp. using DNA from all 88 ileal and88 cecal biopsies showed that the same individuals identified as havinghigh levels of Sutterella by V2 pyrosequencing were also positive by theV6-V8 Sutterella sp.-specific PCR. Additionally, all 4 biopsies perregion in all 7 Sutterella-positive patients showed Sutterellaamplicons, while no amplicons were observed in any Control-GI patientsor ASD-GI patients that lacked Sutterella sequences in V2 pyrosequencingexperiments (FIG. 11). All patients amplicons were confirmed torepresent Sutterella by direct sequencing of PCR products and cloning ofindividual amplicons to create bacterial libraries followed bysequencing of 50 individual clones.

Implementation of Sutterella sp.-specific Real-time PCR forQuantification. Real-time PCR analysis using the same V6-V8 primers anda high coverage Taqman probe (SuttProbel), revealed significantincreases in Sutterella in ASD-GI compared to Control-GI patients forboth the ileum (FIG. 12A: Mann-Whitney U, p=0.0368) and cecum (FIG. 12B:Mann-Whitney U, p=0.0368). Sutterella copy numbers were quite high inboth the ileum and cecum (in the range of 10⁴ to 10⁵ copies) ofSutterella-positive patients (FIG. 12C and FIG. 12D). The distributionof Sutterella abundance by patient and the copy number revealed by V2pyrosequencing and V6-V8 real-time PCR, respectively, were in strikingconcordance (Compare ileum FIG. 8C with FIG. 12C and compare cecum FIG.8D with FIG. 12D). There was 100% congruence between V2 region 454pyrosequencing and both classical and real-time PCR using the V6-V8region Sutterella sp.-specific primers.

Implementation of Sutterella sp.-specific Classical PCR for TaxonomicClassification. Sequences obtained from direct cloning and clonelibraries of the V6-V8 regions of each patient were aligned followingremoval of primer sequences. This analysis revealed that the consensussequence obtained in ileal biopsies matched exactly with sequences incecal biopsies from the same patient. Furthermore, alignment ofsequences revealed that patients 1, 3, 10, 11, and 12 had the exact samesequence for the V6-V8 region, while patients 5 and 7 had a distinct,but identical sequence (FIG. 13). These findings are in agreement withOTU analysis of V2 pyrosequencing reads in which patients 1, 3, 10, 11,and 12 clustered together with OTU 11 containing the majority ofSutterella sequences and patient 5 and 7 clustered together with OTU 38containing the majority Sutterella sequences (FIG. 14). Treeing analysisof the V6-V8 sequences revealed that Sutterella sp. found in patients1,3, 10, 11, and 12 were phylogenetically most closely associated withthe isolates Sutterella stercoricanis (supported by a bootstrapresampling value of 70%) and Parasutterella sp. (supported by abootstrap resampling value of 68%). In contrast, treeing analysisrevealed that Sutterella sp. sequences found in patients 5 and 7 weremost closely associated with the isolate Sutterella wadsworthensis(supported by a bootstrap resampling value of 94%) (FIG. 15A). Thesefindings were consistent with treeing analysis obtained from V2sequences obtained from pyrosequencing analysis in which V2 Sutterellasequences from patients 1,3, 10, 11, and 12 were most closely associatedwith the isolates Sutterella stercoricanis and Sutterella sanguinus(supported by a bootstrap resampling value of 67%) while the V2Sutterella sequences from patients 5 and 7 were most closely associatedwith the isolates of Sutterella wadsworthensis (supported by a bootstrapresampling value of 100%) (FIG. 15B). Thus, sequences from patients 5and 7 clustered with Sutterella wadsworthensis isolates using both theV2 pyrosequencing reads and the V6-V8 sequences obtained from thisassay. In contrast, sequences from patients 1, 3, 10, 11, and 12clustered with Sutterella stercoricanis using both the V2 pyrosequencingreads and the V6-V8 sequence obtained from this assay. However, therewas some divergence between the V2 and V6-V8 regions in determiningrelationships to other isolates (i.e. relatedness to Sutterellasanguinus from the V2 sequences and relatedness to Parasutterella sp.from the V6-V8 sequences).

REFERENCES

-   A1.) Wexler H M, Reeves D, Summanen P H, Molitoris E, McTeague M,    Duncan J, Wilson K H, Finegold S M. 1996. Sutterella wadsworthensis    gen. nov., sp. nov., bile-resistant microaerophilic Campylobacter    gracilis-like clinical isolates. Int J Syst Bacteriol, 46(1):    252-258.-   A2.) Mangin I, Bonnet R, Seksik P, Rigottier-Gois L, Sutren M,    Bouhnik Y, Neut C, Collins M D, Colombel J F, Marteau P,    Dore J. 2004. Molecular inventory of faecal microflora in patients    with Crohn's disease. FEMS Microbiol Ecol, 50(1): 25-36.-   A3.) Gophna U, Sommerfeld K, Gophna S, Doolittle W F, Veldhuyzen van    Zanten S J. 2006. Differences between tissue-associated intestinal    microfloras of patients with Crohn's disease and ulcerative colitis.    J Clin Microbiol, 44(11): 4136-4141.-   A4.) Greetham H L, Collins M D, Gibson G R, Giffard C, Falsen E,    Lawson P A. 2004. Sutterella stercoricanis sp. nov., isolated from    canine faeces. Int J Syst Evol Microbiol. 54: 1581-1584.-   A5.) J Scupham A, Patton T G, Bent E, Bayles D O. 2008. Comparison    of the cecal microbiota of domestic and wild turkeys. Microb Ecol.    56: 322-331.-   A6.) Frank D N, St Amand A L, Feldman R A, Boedeker E C, Harpaz N,    Pace N R. 2007. Molecular-phylogenetic characterization of microbial    community imbalances in human inflammatory bowel diseases. Proc Nati    Acad Sci USA. 104: 13780-13785.-   King A, Downes J, Nord C E, Phillips I; European Study Group. 1999.    Antimicrobial susceptibility of non-Bacteroides fragilis group    anaerobic Gram-negative bacilli in Europe. Clin Microbiol Infect. 5:    404-416.-   Goldstein E J, Citron D M. 2009. Activity of a novel carbapenem,    doripenem, against anaerobic pathogens. Diagn Microbiol Infect Dis.    63: 447-454.-   Wexler H M, Molitoris D, St John S, Vu A, Read E K, Finegold    S M. 2002. In vitro activities of faropenem against 579 strains of    anaerobic bacteria. Antimicrob Agents Chemother. 46: 3669-3675.-   Wexler H M, Molitoris D, Finegold S M. 2000. In vitro activities of    MK-826 (L-749,345) against 363 strains of anaerobic bacteria.    Antimicrob Agents Chemother. 44: 2222-2224.-   Molitoris E, Wexler H M, Finegold S M. 1997. Sources and    antimicrobial susceptibilities of Campylobacter gracilis and    Sutterella wadsworthensis. Clin Infect Dis. Suppl 2: S264-265.-   Wexler H M, Molitoris E, Molitoris D, Finegold S M. 1996. In vitro    activities of trovafloxacin against 557 strains of anaerobic    bacteria. Antimicrob Agents Chemother. 40: 2232-2235.

Example 3 Impaired Carbohydrate Digestion and Transport and MucosalDysbiosis

Gastrointestinal disturbances are commonly reported in children withautism, complicate clinical management, and can contribute to behavioralimpairment. Reports of deficiencies in disaccharidase enzymatic activityand of beneficial responses to probiotic and dietary therapies led tothe survey gene expression and the mucoepithelial microbiota inintestinal biopsies from children with autism and gastrointestinaldisease and children with gastrointestinal disease alone. Ilealtranscripts encoding disaccharidases and hexose transporters weredeficient in children with autism, indicating impairment of the primarypathway for carbohydrate digestion and transport in enterocytes.Deficient expression of these enzymes and transporters was associatedwith expression of the intestinal transcription factor, CDX2.Metagenomic analysis of intestinal bacteria revealed compositionaldysbiosis manifest as decreases in Bacteroidetes, increases in the ratioof Firmicutes to Bacteroidetes, and increases in Betaproteobacteria.Expression levels of disaccharidases and transporters were associatedwith the abundance of affected bacterial phylotypes. These resultsindicate a relationship between human intestinal gene expression andbacterial community structure and provide insights into thepathophysiology of gastrointestinal disturbances in children withautism.

Autism spectrum disorders (ASD) are defined by impairments in verbal andnon-verbal communication, social interactions, and repetitive andstereotyped behaviors. In addition to these core deficits, theprevalence of gastrointestinal (GI) symptoms ranges widely inindividuals with ASD, from 9 to 91% in different study populations [1].Macroscopic and histological observations in ASD include findings ofileo-colonic lymphoid nodular hyperplasia, enterocolitis, gastritis andesophagitis [2,3,4,5,6,7]. Associated changes in intestinal inflammatoryparameters include higher densities of lymphocyte populations, aberrantcytokine profiles, and deposition of immunoglobulin (IgG) and complementC1q on the basolateral enterocyte membrane [5,8,9,10,11,12]. Reportedfunctional disturbances include increased intestinal permeability [13],deficient enzymatic activity of disaccharidases [7], increasedsecretin-induced pancreatico-biliary secretion [7], and abnormalClostridia taxa [14,15,16]. Some children placed on exclusion diets ortreated with the antibiotic vancomycin are reported to improve incognitive and social function [17,18]. Furthermore, a strong correlationbetween GI symptoms and autism severity was found [19].

The intestinal mucoepithelial layer must maximize nutritional uptake ofdietary components while maintaining a barrier to toxins and infectiousagents. Although some aspects of these functions are host-encoded,others are acquired through symbiotic relationships with microbialflora. Dietary carbohydrates enter the intestine as monosaccharides(glucose, fructose, and galactose), disaccharides (lactose, sucrose,maltose), or complex polysaccharides. Following digestion with salivaryand pancreatic amylases, carbohydrates are further digested bydisaccharidases expressed by absorptive enterocytes in the brush borderof the small intestine and transported as monosaccharides across theintestinal epithelium. Although humans lack the glycoside hydrolases andpolysaccharide lyases necessary for cleavage of glycosidic linkagespresent in plant cell wall polysaccharides, oligosaccharides, storagepolysaccharides, and resistant starches, intestinal bacteria encodingthese enzymes expand the capacity to extract energy from dietarypolysaccharides [20,21]. As an end product of polysaccharidefermentation, bacteria produce short-chain fatty acids (butyrate,acetate, and propionate) that serve as energy substrates forcolonocytes, modulate colonic pH, regulate colonic cell proliferationand differentiation, and contribute to hepatic gluconeogenesis andcholesterol synthesis [22,23]. Intestinal microbes also mediatepostnatal development of the gut mucoepithelial layer, provideresistance to potential pathogens, regulate development ofintraepithelial lymphocytes and Peyer's patches, influence cytokineproduction and serum immunoglobulin levels, promote systemic lymphoidorganogenesis, and influence brain development and behavior [24,25,26].

Although bacteria have been examined in fecal material from childrenwith autism, no study to date has reported analyses of microbiotaadherent to their intestinal mucoepithelium. Furthermore, there are noreports wherein intestinal gene expression in children with autism hasbeen correlated with alterations in intestinal microbiota. GIdysfunction is commonly reported in children with autism; however, itremains unclear how or whether GI dysfunction in children with autismdiffers from GI dysfunction found in typically developing children.Expression of human genes involved in carbohydrate digestion andtransport was investigated along with bacterial community composition inintestinal biopsies from children with autistic disorder and GI disease(AUT-GI) compared to children with GI disease alone (Control-GI).Results from gene expression assays and metagenomic analysis of overhalf a million bacterial 16S rRNA gene sequences revealed decreased mRNAexpression for human disaccharidases and hexose transporters andcompositional dysbiosis in children in the AUT-GI group compared tothose in the Control-GI group. Results described herein show the complexrelationship between human intestinal gene expression and bacterialcommunity structure, and provide insights into the molecular mechanismsunderlying the pathophysiology of gastrointestinal disturbances inchildren with autism.

Results

Patient Characteristics

All AUT-GI and Control-GI children evaluated were male (Table 6A). Meanonset age for autism in AUT-GI was 13.4+/−5.4 months. Median age atbiopsy was similar for AUT-GI and Control-GI children [median age inyears (interquartile range, IQR), AUT-GI, 4.5 (1.3); and Control-GI, 4.0(1.1)]. Median number of medications used and the IQR for number ofmedications used per subject were identical in AUT-GI and Control-GIchildren. Food allergies (FA) were commonly reported in both AUT-GI(67%) and Control-GI (71%) subjects. The majority of children with FAhad reported milk-related allergy (90% for AUT-GI and 100% forControl-GI) and/or wheat-related allergy (80% for AUT-GI and 80% forControl-GI). Beneficial effects of dietary intervention on GIdisturbances were reported for all AUT-GI and Control-GI subjects withFA. Comorbid conditions were reported in 67% of AUT-GI children and 100%of Control-GI children. The most commonly reported comorbid conditionswere atopic manifestations (asthma, atopic dermatitis, and allergicrhinitis). Atopic manifestations were more common in Control-GI children(100%) than AUT-GI children (53%) (Table 6A). The frequency ofindividual atopic manifestations was higher in Control-GI children. Thelargest difference in frequency was for asthma, which was only reportedin 20% of AUT-GI children compared to 71% of Control-GI children (Table6A). Established intestinal disorders were only reported in a fewsubjects: two AUT-GI subjects (13%: 1 with IBD, 1 with Celiac disease)and one Control-GI subject (14%: IBD). For detailed information relatedto medication use, food allergy and comorbid conditions in individualAUT-GI and Control-GI children see Table 7. The prevalence of specificGI symptoms was similar in AUT-GI and Control-GI children (Table 6B).The most frequently reported GI symptoms in both groups were diarrhea(AUT-GI, 80%; Conrol-GI, 71%) and changes in stool frequency (AUT-GI,87%; Control-GI, 71%) and consistency (AUT-GI, 80%; Control-GI, 86%).Mucus in stool was more frequent in Control-GI (86%) compared to AUT-GI(40%) children; bloating was more frequent in AUT-GI (60%) compared toControl-GI (29%) children. Regression (loss of language and/or otherskills following acquisition) is reported in 20% to 40% of individualswith autism, and some studies indicate higher rates of GI symptoms inASD subjects with regression than those without regression [27]. 87% ofthe AUT-GI subjects had behavioral regression (Table 8).

TABLE 6A, B Summary of patient characteristics. Control- AUT-GI GISubject Characteristic Subcategory (n = 15) (n = 7) Autism onset age inmonths, AUT-GI subjects 13.4 ± 5.4 — mean ± SD Gender All subjects Allmale All male Ethnicity, n (%) Caucasian 14 (93)  6 (86) Hispanic 1 (7) 0 (0)  African-American 0 (0)  1 (14) Age at biopsy in years, Allsubjects 4.5 (1.3)  4.0 (1.1)  median (IQR) [range] [3.5-5.9] [3.9-5.5]Medications-number per All subjects 5 (7)  5 (7)  subject^(a), median(IQR)  [1-21] [0-8] [range] Food allergies, n (% of All subjects 10(67)  5 (71) subjects) Milk-related allergy^(b), n (% Subjects reporting9 (90)  5 (100) of subjects with food allergy) any food allergyWheat-related allergy^(c), n (% Subjects reporting 8 (80) 4 (80) ofsubjects with food allergy) any food allergy Diet improvement of GISubjects reporting 10 (100)  5 (100) problems, n (% of subjects any foodallergy with food allergy) Current comorbid All subjects   1 (1.75)   2(2.75) conditions-number per [0-5] [1-6] subject, median (IQR) [range]Comorbid atopic disease All subjects 8 (53)  7 (100) manifestations^(d),n (% of subjects) Asthma, n (% of subjects) All subjects 3 (20) 5 (71)Atopic dermatitis, n (% of All subjects 4 (27) 4 (57) subjects) Allergicrhinitis, n (% of All subjects 4 (27) 3 (43) subjects) ^(a)Number ofprescription drugs and alternative agents taken regularly, per subject^(b)Allergy to milk, casein, lactose or dairy ^(c)Allergy to wheat orgluten ^(d)Asthma, Allergic rhinitis, or Atopic dermatitis

TABLE 6B Summary of patients' GI symptoms. GI Symptoms AUT-GI, n (%)Control-GI, n (%) Diarrhea 12 (80)  5 (71) Diarrhea w/ Vomiting 2 (13) 2(29) Vomiting 2 (13) 1 (14) Bloating 9 (60) 2 (29) Δ Stool Frequency 13(87)  5 (71) Δ Stool Consistency 12 (80)  6 (86) Mucus in Stool 6 (40) 6(86) Blood in Stool 2 (13) 1 (14) Pain 8 (53) 5 (71) Weight Loss 3 (20)0 (0)  Fever 1 (7)  0 (0) 

TABLE 7 Reported comorbid conditions, food allergies, and medication useby patient Current Comorbid Food Allergy Patient # Group ConditionsReported Medications 1 AUT-GI asthma, atopic milk, gluten, Vitamin B1,B2, B3, B6, B9, dermatitis, celiac eggs, B12, C, E; Ca, Zn, Fish oil,disease, movement peanuts, tree Omega-3-fatty acids, disorder, myopathynuts, soy, Probiotic, Ibuprofen, corn, peas Lanzoprazole, Montelukastsodium, Levalbuterol inhaler, Albuterol inhaler 2 AUT-GI allergicrhinitis milk, gluten, Vitamin C; MVM, Ca/Mg eggs supplement, Omeprazole3 AUT-GI IBD Milk, gluten, Vitamin B12, C; MVM, dyes Ca/Mg supplement,Zn, flaxseed oil, antifungal herbal agent, digestive enzymes 4 AUT-GIallergic rhinitis, casein, gluten Vitamin A, C, Methyl-B12, asthma,atopic Folinic acid; MVM, Ca/Mg dermatitis, migraine supplement, Zn, Mb,Fish oil, Omega-3-fatty acids, SAMe, Inositol, Selenomethionine,Trimethylglycine, 5- methyl-tetrahydrofolate, Transdermal glutathione,MgSO4 cream, Zn soy cream, DMAE, DMPS, Alpha lipoic acid, Montelukastsodium 5 AUT-GI atopic dermatitis lactose MVM 6 AUT-GI allergicrhinitis, gluten, corn, Vitamin D; Ca, Zn, Mg, P, frequent URI, soyFlaxseed oil, Probiotic, epilepsy Artichoke extract, Sarsaparillaextract, Wasabi powder, Lipase, Amylase, Protease 7 AUT-GI allergicrhinitis, milk, gluten, Folinic acid; MVM, Ca/Mg frequent otitis sweetsupplement, media potatoes, Trimethylglycine, Lipase, oranges, Amylase,Protease, berries Cellulase, Lactase 8 AUT-GI none none reported VitaminB complex, L- carnitine, Lipase, Amylase, Protease, Diphenhydramine,Acetaminophen, Ibuprofen, Melatonin, Sertraline, Valproic acid 9 AUT-GInone none reported MVM, Ca 10 AUT-GI none none reported Omeprazole 11AUT-GI atopic dermatitis cow's milk, Flaxseed oil, Coenzyme goat's milk,Q10, Cell signal barley, enhancers (CSE-14, 15), carrots, Probiotic,Lipase bananas, cantelope, coffee, cranberry, lamb, lettuce 12 AUT-GIEpstein-Barr virus dairy, wheat, Methyl-B12, DMSA, infectionsalicylates, Amphoterecin B Phenols 13 AUT-GI asthma dairy, wheat,Vitamin B12; Ca/Mg yeast supplement, Zn, Probiotic, Clonidine, Secretin14 AUT-GI none none reported MVM, F 15 AUT-GI none none reported Lipase,Amylase, Protease, Milk of magnesia, Lansoprazole 16 Control- allergicrhinitis, none reported MVM, Montelukast sodium, GI asthma, atopicFluticasone propionate, dermatitis, frequent Lansoprazole, Amoxicillinsinusitis 17 Control- atopic dermatitis none reported Ca citrate,Mg/amino acid GI complex, Hydroxyzine, Budesonide, Prednisolone,Montelukast sodium, Levalbuterol inhaler, Tacrolimus 18 Control- asthmadairy, peanuts Ibuprofen GI 19 Control- asthma, atopic milk, wheat,Lipase, Amylase, Protease, GI dermatitis, IBD, eggs, oats,Diphenhydramine, Cetirizine dysphagia, salmon, soy, hydrochloride,Omeprazole, microcytic anemia, peanut, tree Budesonide, Montelukastpancreatic nut, chicken, sodium, Levalbuterol inhaler insufficiencyturkey, beef, broccoli, cabbage, lentils, legumes 20 Control- allergicrhinitis, dairy, gluten, Vitamin B12, Fish oil, Milk GI asthma, atopiceggs, soy, thistle, DMSA, Allithiamine dermatitis citrus 21 Control-asthma dairy, wheat, Probiotic GI eggs, fruit 22 Control- allergicrhinitis, dairy, wheat, none reported GI vitiligo eggs, peanuts, beefIBD—Inflammatory Bowel Disease; URI—Upper respiratory tract infection;MVM—multivitamin with minerals; SAMe—S-adenosylmethionine;DMAE—dimethylaminoethanol; DMPS—2,3-Dimercapto-1-propanesulfonic acid;DMSA—Dimercaptosuccinic acid

TABLE 8 Reported behavioral regression in AUT-GI children. AUT/GI cases(n = 15) PHENOTYPIC CHARACTERISTICS n (%) ANY REPORTED LOSS (ADI-R orCDI) 13 (87) ADI-R LOSS Language loss 11 (73) ITEMS Other skill loss 12(80) (with or without language loss) Other skill loss  2 (13) withoutlanguage loss CPEA Word loss regression 12 (80) REGRESSION Non-word lossregression 1 (7) CATEGORY No regression  2 (13) Legend: ADI-R, AutismDiagnostic Interview-Revised; CDI, MacArthur Communicative DevelopmentInventory; CPEA, Collaborative Program for Excellence in Autism.

Deficient Ileal mRNA Expression of Disaccharidases and HexoseTransporters in AUT-GI Children

Transcript levels were examined for three primary brush borderdisaccharidases (sucrase isomaltase [S1], maltase glucoamylase [MGAM],and lactase [LCT]) in ileal biopsies of AUT-GI and Control-GI childrenby real time PCR. Levels of mRNA for all three enzymes were decreased inAUT-GI children: SI (FIG. 16A: Mann-Whitney, p=0.001), MGAM (FIG. 16B:Mann-Whitney, p=0.003) and LCT (FIG. 16C: Mann-Whitney, p=0.032). Withinthe AUT-GI group, 86.7%, 80%, and 80% of children had deficienttranscript levels (defined as below the 25^(th) percentile of valuesobtained for Control-GI children and at least two-fold below Control-GImean values) for SI, MGAM, and LCT, respectively (Table 9A and Table10). Nearly all (14/15, or 93.3%) AUT-GI children had deficiencies in atleast one disaccharidase enzyme; 80% had deficiencies in 2 or moreenzymes; 73.3% had deficiencies in all three enzymes (Table 9A).Deficiencies in LCT mRNA in AUT-GI children were not attributable todisproportionate adult-type hypolactasia genotypes in the AUT-GI grouprelative to the Control-GI group (FIG. 36A-D).

TABLE 9 Patient summary tables for gene expression and bacterial assays.

(A-C) Legend: Increases or decreases in AUT-GI children in both geneexpression (A) and bacterial parameters (B and C) were determined foreach individual based on the levels of each parameter in the Control-GIgroup. (A) The gene expression levels in the AUT-GI children thatexceeded the 75^(th) percentile of Control-GI values and were at least2-fold increased relative to the Control-GI mean (arrow pointing up) orbelow the 25^(th) percentile of Control-GI values and at least 2-folddecreased relative to the Control-GI mean (arrow pointing down) werescored as an increase or decrease, respectively. (B and C) Bacterialparameters in AUT-GI children that exceeded the 75^(th) percentile ofControl-GI values (arrows pointing up) or were below the 25^(th)percentile of Control-GI values (arrows pointing down) were scored as anincrease or decrease, respectively. Values above the 90^(th) or belowthe 10^(th) percentiles of Control-GI children are indicated by doublearrows. Results are shown for data obtained by real-time PCR (RT), whereperformed, and pyrosequencing (454). (n.c. = no change relative todefined cut-off values for Control-GI children).

TABLE 10 Fold-change in gene expression in AUT-GI children.

Legend: Fold-change values were calculated realtive to the meanexpression level obtained for all Control-GI children for each gene.Expression levels for individual patients that were at least 2-foldincreased (>2) or decreased (<0.5) relative to the Control-GI mean(grey*) are highlighted in gray, and dark gray, respectively.

Two hexose transporters, sodium-dependent glucose cotransporter (SGLT1)and glucose transporter 2 (GLUT2), mediate transport of monosaccharidesin the intestine. SGLT1, located on the luminal membrane of enterocytes,is responsible for the active transport of glucose and galactose fromthe intestinal lumen into enterocytes. GLUT2 transports glucose,galactose and fructose across the basolateral membrane into thecirculation and can also translocate to the apical membrane [28].Real-time PCR revealed a decrease in ileal SGLT1 mRNA (FIG. 16D:Mann-Whitney, p=0.008) and GLUT2 mRNA (FIG. 16E: Mann-Whitney, p=0.010)in AUT-GI children. For SGLT1, 73.3% of AUT-GI children had deficienttranscript levels; 73.3% of AUT-GI children had deficient GLUT2transcript levels relative to Control-GI children (Table 9A).Deficiencies were found in at least one hexose transporter in 80% ofAUT-GI children; 66.7% had deficiencies in both transporters.

In total, 93.3% (14/15) of AUT-GI children had mRNA deficiencies in atleast one of the 5 genes involved in carbohydrate digestion ortransport; 66.7% (10/15) had mRNA deficiencies in all 5 genes (Table9A).

To determine whether reductions in disaccharidase and transportertranscript levels reflected loss of or damage to intestinal epithelialcells, mRNA levels associated with a tissue-specific marker restrictedto these cells, villin [29,30] was measured. Ileal villin mRNA levelswere not decreased in AUT-GI children (Mann-Whitney, p=0.307) (FIG.16F). Normalization of SI, MGAM, LCT, SGLT1 and GLUT2 to villin mRNAlevels did not correct deficits (FIG. 22A-E).

The transcription factor, caudal type homeobox 2 (CDX2), regulatesexpression of SI, LCT, GLUT2, and SGLT1 [31,32,33,34]. Real-time PCRexperiments demonstrated lower levels of CDX2 mRNA in some AUT-GIsubjects versus controls; however, group differences were notsignificant (FIG. 16G: Mann-Whitney, p=0.192). Although only 33.3% ofAUT-Gl patients had deficient CDX2 mRNA levels (Table 9A), 86.7% ofAUT-GI children had CDX2 levels below the 50^(th) percentile ofControl-GI children and 46.7% of AUT-GI children had at least a two-folddecrease in CDX2 expression relative to the Control-GI mean. Only oneAUT-GI child (patient #7) had CDX2 levels above the 75^(th) percentileof Control-GI children and a near 2-fold (1.95-fold) increase in CDX2expression (Table 9A and Table 10). This child was the only AUT-GIsubject who did not show signs of deficiencies in disaccharidases ortransporters.

AUT-GI children with deficiencies in all five disaccharidases andtranporters had significantly lower levels of CDX2 mRNA compared toAUT-GI children with fewer than five deficiencies (FIG. 33:Mann-Whitney, p=0.037). However, only a trend toward decreased CDX2levels was found when comparing AUT-GI children with deficiencies in allfive disaccharidases and transporters and Control-GI children (FIG. 33:Mann-Whitney, p=0.064).

Multiple linear regression analysis was conducted to determine whetherdiagnostic status (AUT-GI or Control-GI), CDX2 mRNA expression, orvillin mRNA expression (predictor variables) was associated with mRNAexpression levels of individual disaccharidases (SI, MGAM, LCT) ortransporters (SGLT1, GLUT2) (Table 11). In each of the five models,where the expression of SI, MGAM, LCT, SGLT1, or GLUT2 served as outcomevariables, CDX2 contributed significantly to the model. As the level ofCDX2 increased by one unit of standard deviation, there was aconcomitant approximate one unit increase in log-transformeddisaccharidase and transporter transcript levels (ranging from 0.78 forSGLT1 to 1.30 for LCT). None of the interaction terms between CDX2 andstatus were significant, indicating that the magnitude of the effect ofCDX2 on log-transformed enzyme and transporter levels was the same forAUT-GI and Control-GI children. For SGLT1 and GLUT2 expression, CDX2 wasthe sole significant predictor variable in the model. Status and CDX2were significant predictors of SI, MGAM, and LCT expression, indicateingthat additional factors associated with status must also contribute toexpression levels for these enzymes. Villin was not a significantpredictor of the expression levels of any of the five genes afteradjusting for CDX2.

TABLE 11 Multiple linear regression analysis examining CDX2 and villinas predictors of disaccharidase and transporter mRNA expression amongAUT-GI and Control-GI children. Ad- Predictor Variables: OutcomeF_(3,18) justed Coefficient Estimate Variable (p-value) R² StatusCDX2^(STDev) Villin^(STDev) SI 10.35 0.57 −1.83* 0.93* −0.19 (0.0003)***MGAM 8.78 0.53 −2.10* 1.15* −0.20 (0.0008)*** LCT 10.87 0.59 −2.25*1.30* 0.65 (0.0003)*** SGLT1 6.88 0.46 −1.36† 0.78* 0.12 (0.0030)**GLUT2 6.06 0.42 −1.90† 1.06* 0.03 (0.0050)** ^(STDev)Change inlog-transformed outcome variable levels per unit standard deviationincrease in predictor variable *p < 0.05; **p < 0.01; ***p < 0.001; †p <0.1 (trend)

Mucosal Dysbiosis in AUT-GI Children

To determine whether deficient carbohydrate digestion and absorptioninfluenced the composition of intestinal microflora, ileal and cecalbiopsies from AUT-GI and Control-GI children were analyzed by bacterial16S rRNA gene pyrosquencing. The use of biopsies rather than fecalmaterial allowed us to assess the mucoepithelia-associated microbiota,as these likely establish more intimate interactions with the humanintestinal epithelium and immune cells [35]. A total of 525,519bacterial sequences were subjected to OTU (Operational Taxonomic Unit;defined at 97% identity) analysis and classified with RDP (RibosomalDatabase Project). Rarefaction analysis of OTUs did not indicate a lossor gain of overall diversity based on Shannon Diversity estimates inAUT-GI compared to Control-GI children (See FIG. 23A-D).

Classification of pyrosequencing reads revealed that Bacteroidetes andFirmicutes were the most prevalent taxa in ileal and cecal tissues ofAUT-GI and Control-GI children, followed by Proteobacteria (FIG. 17A,B). Other phyla identified at lower levels included Verrucomicrobia,Actinobacteria, Fusobacteria, Lentisphaerae, and TM7, as well as“unclassified bacteria” (sequences that could not be assigned at thephylum-level) (FIG. 17A, B). The abundance of Bacteroidetes was lower inAUT-GI ileal (FIG. 17C: Mann-Whitney, p=0.012) and cecal biopsies (FIG.17D: Mann-Whitney, p=0.008) as compared with the abundance ofBacteroidetes in Control-GI biopsies. Real-time PCR usingBacteroidete-specific primers confirmed decreases in Bacteroidetes inAUT-GI ilea (FIG. 17E: Mann-Whitney, p=0.003; Table 12: 50% averagereduction in Bacteroidete 16S rDNA copies; range, 24.36% to 76.28%decrease) and ceca (FIG. 17F: Mann-Whitney, p=0.022; Table 12: 29%average reduction in 13 of 15 patients with reduced Bacteroidetes;range, 7.22% to 56.54% decrease), with levels below the 25^(th)percentile of Control-GI children in 100% of AUT-GI ilea and 86.7% ofAUT-GI ceca (Table 9B). OTU analysis of Bacteroidete sequencesindicateed that deficiencies in Bacteroidete sequences in AUT-GIsubjects were attributable to cumulative losses of 12 predominantphylotypes of Bacteroidetes, rather than loss of any one specificphylotype (FIG. 25A-E).

TABLE 12 Percent change in bacterial levels in AUT-GI children.Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes FirmicutesFirmicutes Firmicutes Firmicutes Patient RT—Ileum RT—Cecum 454—Ileum454—Cecum RT—Ileum RT—Cecum 454—Ileum 454—Cecum # % Change % Change %Difference % Difference % Change % Change % Difference % Difference 1−38.45 −45.21 −12.97 −8.04 22.53 39.63 12.22 8.24 2 −76.28 −32.49 −41.41−18.38 57.67 96.65 −6.97 1.43 3 −54.81 −27.47 −9.17 −13.61 26.31 86.516.54 7.84 4 −61.97 −16.71 −18.16 −11.57 132.28 77.63 17.25 13.88 5−48.68 −22.27 5.65 3.02 5.58 −3.18 −5.16 −5.10 6 −38.60 0.94 0.80 4.7719.05 131.95 5.23 −0.68 7 −38.80 −14.12 −4.85 −12.24 −2.18 48.42 10.2314.63 8 −53.67 −50.41 −20.58 −21.64 −13.25 2.35 3.26 3.41 9 −41.25−17.14 −12.58 −10.11 24.21 22.06 16.30 8.73 10 −40.14 −9.41 −10.88−12.04 −13.93 18.59 13.69 12.11 11 −70.52 −55.54 −13.25 −16.26 45.3383.50 3.06 3.85 12 −35.81 −7.22 −0.12 −4.52 17.67 30.06 2.06 3.13 13−47.99 −40.26 −6.34 −20.96 14.14 49.60 8.90 10.92 14 −24.36 13.00 −7.63−3.02 8.86 15.44 12.74 5.19 15 −75.62 −34.87 −29.30 −14.41 −60.76 −62.03−14.79 −16.49 Lach. + Lach. + Proteo- Proteo- Beta- Beta- ClostridiaClostridia Rumino. Rumino. bacteria bacteria Proteoabact. Proteoabact.Patient 454—Ileum 454—Cecum 454—Ileum 454—Cecum 454—Ileum 454—Cecum454—Ileum 454—Cecum # % Difference % Difference % Difference %Difference % Difference % Difference % Difference % Difference 1 13.338.84 14.51 9.92 1.52 0.82 4.36 3.00 2 −6.26 1.56 −6.60 1.87 47.77 16.2324.82 7.16 3 7.34 8.11 5.45 6.39 −0.57 1.95 3.10 3.12 4 17.72 14.2018.72 15.29 1.90 −1.27 1.55 0.02 5 −4.24 −4.49 −3.18 −3.61 −0.02 2.724.46 3.99 6 6.14 0.06 5.81 0.35 −5.00 −2.99 −0.66 −0.78 7 10.91 15.1712.31 16.49 −4.34 −1.26 0.17 0.89 8 3.04 2.34 3.97 3.24 18.40 19.27−1.35 −0.42 9 17.34 9.46 17.21 9.35 −3.27 1.85 0.54 2.69 10 14.97 12.7815.52 13.44 −1.98 0.73 2.43 2.79 11 3.91 4.30 3.85 4.18 8.05 10.52 5.556.16 12 2.73 3.56 2.05 2.15 −1.82 1.25 2.34 2.90 13 9.86 11.17 10.4711.74 −3.60 8.33 0.06 4.70 14 13.66 6.03 15.60 7.61 −4.09 −1.09 −0.58−0.15 15 −15.60 −16.02 −14.36 −14.15 44.83 31.90 −0.43 −0.79 Firm./Firm./ Firm./ Firm./ Clostrid./ Clostrid./ Bacteroid. Bacteroid.Bacteroid. Bacteroid. Bacteroid. Bacteroid. Firm. + Firm. + Ratio RatioRatio Ratio Ratio Ratio Proteobact. Proteobact. Patient RT—IleumRT—Cecum 454—Ileum 454—Cocum 454—Ileum 454—Cecum 454—Ileum 454—Cecum # %Change % Change % Change % Change % Change % Change % Change % Change 185.85 130.48 80.63 48.14 89.92 52.85 13.73 9.06 2 520.47 163.47 81.0439.53 83.47 39.47 40.79 17.66 3 160.98 132.59 43.00 60.37 47.41 62.465.97 9.79 4 470.19 92.89 126.70 84.43 133.22 87.82 19.15 12.62 5 92.0912.65 −28.53 −26.23 −26.69 −25.13 −5.18 −2.38 6 81.61 107.84 17.08−10.99 21.09 −8.72 0.24 −3.67 7 49.24 56.32 48.85 90.21 53.38 94.97 5.8913.37 8 74.81 86.66 59.00 60.28 57.63 53.77 21.65 22.68 9 97.40 33.2499.17 55.50 107.51 59.77 13.04 10.59 10 34.24 18.40 81.88 77.16 90.5082.97 11.91 12.84 11 360.23 281.88 36.65 46.99 40.77 49.43 11.11 14.3712 71.17 26.80 6.13 18.04 8.99 20.18 0.24 4.39 13 104.90 126.51 46.68101.59 55.16 107.58 5.30 19.25 14 34.37 −7.59 66.89 24.23 74.34 28.288.65 4.09 15 50.31 −47.27 −30.42 −62.02 −43.09 −63.48 30.04 15.41Legend: Percent change values were calculated for real-time PCR andratio data relative to the mean levels obtained for all Control-GIchildren for each bacterial variable. Percent difference values werecalculated for pyrosequencing data by subtracting the mean percentabundance of Control-GI children from the percent abundance of eachAUT-GI patient for each variable.

Analysis of pyrosequencing reads revealed a significant increase inFirmicute/Bacteroidete ratios in AUT-GI ilea (FIG. 18A: Mann-Whitney,p=0.026) and ceca (FIG. 18B: Mann-Whitney, p=0.032). An increase wasalso observed at the order level for Clostridiales/Bacteroidales ratiosin ilea (FIG. 26A: Mann-Whitney, p=0.012) and ceca (FIG. 26B:Mann-Whitney, p=0.032). Real-time PCR using Firmicute- andBacteroidete-specific primers confirmed increases inFirmicute/Bacteroidete ratios in AUT-GI ilea (FIG. 30C: Mann-Whitney,p=0.0006) and ceca (FIG. 30D: Mann-Whitney, p=0.022). Based on real-timePCR results, Firmicute/Bacteroidete ratios were above the 75^(th)percentile of Control-GI values in 100% of AUT-GI ilea and 60% of AUT-GIceca (Table 9C).

The cumulative level of Firmicutes and Proteobacteria was significantlyhigher in the AUT-GI group in both ileal (FIG. 18G: Mann-Whitney,p=0.015) and cecal (FIG. 18H: Mann-Whitney, p=0.007) biopsies; however,neither Firmicute nor Proteobacteria levels showed significantdifferences on their own (FIG. 27A-D and FIG. 19A, B). These resultsindicate that the observed decrease in Bacteroidetes in AUT-GI childrenis accompanied by an increase in Firmicutes (Ileal biopsies—Patients 1,3, 4, 6, 7, 9, 10, 13, and 14; Cecal biopsies—Patients 1, 3, 4, 7, 9,10, and 13), or Proteobacteria (Ileal biopsies—Patients 2, 8, 11 and 15;Cecal biopsies—Patients 2, 5, 8, 11, 13, and 15), or both (Cecalbiopsies—Patient 13) (Table 9B and FIG. 34A-B).

Within the Firmicute phyla, order-level analysis of pyrosequencing readsindicated trends toward increases in Clostridiales in AUT-GI ilea (FIG.27E: Mann-Whitney, p=0.072) and ceca (FIG. 27F: Mann-Whitney, p=0.098).Family-level analysis revealed that increased Clostridiales levels inAUT-GI patient samples were largely attributable to increases inLachnospiraceae and Ruminococcaceae (FIG. 18C-F). Cumulative levels ofLachnospiraceae and Ruminococcaceae above the 75^(th) percentile of thecorresponding levels in Control-GI samples were found in 60% of AUT-GIileal and 53.3% of AUT-GI cecal samples (Table 9B). Genus-level analysisindicated that members of the genus Faecalibacterium within the familyRuminococcaceae contributed to the overall trend toward increasedClostridia levels (FIG. 28A-B). Within Lachnospiraceae, members of thegenus Lachnopsiraceae Incertae Sedis, Unclassified Lachnospiraceae, andto a lesser extent Bryantella (cecum only), contributed to the overalltrend toward increased Clostridia (FIG. 28A-B).

Within the Proteobacteria phyla, levels of Betaproteobacteria tended tobe higher in the ilea of AUT-GI patients (FIG. 19C: Mann-Whitney,p=0.072); significantly higher levels of Betaproteobacteria were foundin AUT-GI ceca (FIG. 19D: Mann-Whitney, p=0.038). Levels ofBetaproteobacteria were above the 75^(th) percentile of Control-GIchildren in 53.3% of AUT-GI ilea and 66.7% of AUT-GI ceca (Table 9B).Family-level analysis revealed that members of the familiesAlcaligenaceae (patients #1, 3, 5, 7, 10, 11, and 12) and Incertae Sedis5 (patient #2 only) contributed to the increases in Betaproteobacteriain ilea (FIG. 19E) and ceca (FIG. 19F). Alcaligenaceae sequences weredetected in 46.7% of AUT-GI children and none of the Control-GIchildren. Elevated levels of Proteobacteria in AUT-GI ilea and cecareflected increased Alpha-(families Methylobacteriaceae and UnclassifiedRhizobiales) and Betaproteobacteria (family Incertae Sedis 5) forpatient #2 and increased Gammaproteobacteria (family Enterobacteriaceae)for patients #8 and #15 (FIG. 19E-F). Levels of Alpha-, Delta-, Gamma-,and Epsilonproteobacteria were not significantly different betweenAUT-GI and Control-GI samples.

The use of probiotics, proton-pump inhibitors, or antibiotics has beenshown to impact the intestinal microbiome [36,37,38]. Analysis of thepotential effects of these agents in this cohort revealed only onepotential confounding effect: a correlation between the ratio ofFirmicutes to Bacteroidetes in the cecum obtained by real-time PCR inAUT-GI children who had taken probiotics (Table 13A). No effect ofproton-pump inhibitors was observed for any of the significant variablesassessed in this study (Table 13B). Only one patient, a control(Control-GI patient #16), had taken an antibiotic (amoxicillin) in thethree months prior to biopsy (See Table 13C).

TABLE 13A Evaluation of confounding effects attributed to the use ofprobiotics (Pb). AUT(−Pb) vs. AUT(−Pb) vs. Control(−Pb)^(a),AUT(+Pb)^(b), p-value^(MW), p-value^(MW), [effect in [effect in VariableAUT(−Pb)] AUT(+Pb)] SI 0.007**, [decreased] 0.602, [no change] MGAM0.007**, [decreased] 0.240, [no change] LCT 0.012*, [decreased] 0.695,[no change] SGLT1 0.021*, [decreased] 0.433, [no change] GLUT2 0.021*,[decreased] 0.794, [no change] Bacteroidetes IL(RT) 0.009**, [decreased]0.602, [no change] Bacteroidetes CEC(RT) 0.056†, [decreased] 0.192, [nochange] Bacteroidetes IL(454) 0.035*, [decreased] 0.602, [no change]Bacteroidetes CEC(454) 0.009**, [decreased] 0.999, [no change]Firm./Bacteroid. Ratio 0.004**, [increased] 0.361, [no change] IL(RT)Firm./Bacteroid. Ratio 0.159, [no change] 0.037*, [increased] CEC(RT)Firm./Bacteroid. Ratio 0.070†, [increased] 0.514, [no change] IL(454)Firm./Bacteroid. Ratio 0.056†, [increased] 0.896, [no change] CEC(454)Clostridiales/Bacteroidales 0.044*, [increased] 0.695, [no change]IL(454) Clostridiales/Bacteroidales 0.070†, [increased] 0.896, [nochange] CEC(454) Beta-proteobacteria 0.108, [not significant] 0.361, [nochange] CEC(454) ^(a)AUT(−Pb), n = 11; Control (−Pb), n = 6^(b)AUT(−Pb), n = 11; AUT(+Pb), n = 4 ^(MW)Mann-Whitney test

TABLE 13B Evaluation of confounding effects attributed to use ofproton-pump inhibitors (PPI). AUT(−PPI) vs. AUT(−PPI) vs.Control(−PPI)^(a), AUT(+PPI)^(b), p-value^(MW), p-value^(MW), [effect in[effect in Variable AUT(−PPI)] AUT(+PPI)] SI 0.003**, [decreased] 0.794,[no change] MGAM 0.006**, [decreased] 0.695, [no change] LCT 0.234, [nochange] 0.192, [no change] SGLT1 0.036*, [decreased] 0.896, [no chane]GLUT2 0.036*, [decreased] 0.602, [no change] Bacteroidetes IL(RT)0.002**, [decreased] 0.433, [no change] Bacteroidetes CEC(RT) 0.011*,[decreased] 0.433, [no change] Bacteroidetes IL(454) 0.036*, [decreased]0.050†, [decreased] Bacteroidetes CEC(454) 0.036*, [decreased] 0.514,[no change] Firm./Bacteroid. Ratio 0.004**, [increased] 0.602, [nochange] IL(RT) Firm./Bacteroid. Ratio 0.011*, [increased] 0.896, [nochange] CEC(RT) Firm./Bacteroid. Ratio 0.027*, [increased] 0.514, [nochange] IL(454) Firm./Bacteroid. Ratio 0.036*, [increased] 0.514, [nochange] CEC(454) Clostridiales/Bacteroidales 0.015*, [increased] 0.514,[no change] IL(454) Clostridiales/Bacteroidales 0.036*, [increased]0.514, [no change] CEC(454) Beta-proteobacteria 0.047*, [increased]0.794, [no change] CEC(454) ^(a)AUT(−PPI), n = 11; Control(−PPI), n = 5^(b)AUT(−PPI), n = 11; AUT(+PPI), n = 4 ^(MW)Mann-Whitney test

TABLE 13C Evaluation of confounding effects attributed to the use ofantibiotics. Including Antibiotic Excluding Antibiotic User (Ab) User(Ab) AUT (−Ab) vs. AUT (−Ab) vs. Control (+Ab Control (−Ab)^(b), and−Ab)^(a), p-value^(MW), p-value^(MW), [effect [effect in Variable inAUT(−Ab)] AUT(−Ab)] SI 0.001**, [decreased] 0.003**, [decreased] MGAM0.003**, [decreased] 0.010**, [decreased] LCT 0.032*, [decreased]0.062†, [decreased] SGLT1 0.008**, [decreased] 0.020*, [decreased] GLUT20.010*, [decreased] 0.024*, [decreased] Bacteroidetes IL (RT) 0.003**,[decreased] 0.0005***, [decreased] Bacteroidetes CEC (RT) 0.022*,[decreased] 0.002**, [decreased] Bacteroidetes IL (454) 0.012*,[decreased] 0.005**, [decreased] Bacteroidetes CEC (454) 0.008**,[decreased] 0.008**, [decreased] Firm./Bacteroid. 0.0006***, [increased]0.001**, [increased] Ratio IL (RT) Firm./Bacteroid. 0.022*, [increased]0.008**, [increased] Ratio CEC (RT) Firm./Bacteroid. 0.026*, [increased]0.013*, [increased] Ratio IL (454) Firm./Bacteroid. 0.032*, [increased]0.029*, [increased] Ratio CEC (454) Clostridiales/ 0.012*, [increased]0.008**, [increased] Bacteroidales IL (454) Clostridiales/ 0.032*,[increased] 0.024*, [increased] Bacteroidales CEC (454)Beta-proteobacteria 0.038*, [increased] 0.120, [no change] CEC (454)^(a)AUT(−Ab), n = 15; Control(+Ab and −Ab), n = 7 ^(b)AUT(−Ab), n = 15;Control(−Ab), n = 6 ^(MW)Mann-Whitney test

Disaccharidase and Transporter mRNA Levels as Predictors of BacterialAbundance

Multiple linear regression analysis was conducted to determine whetherdiagnostic status (AUT-GI or Control-GI) and mRNA expression ofdisaccharidases (SI, MGAM and LCT) and transporters (SGLT1 and GLUT2)(predictor variables) were associated with bacterial levels as outcomevariables (Table 14). For Bacteroidetes, SGLT1 (ileum and cecum) and SI(cecum only) were significant predictors. In both the ileum and cecum,Bacteroidete levels increased as SGLT1 transcript levels increased. Inthe cecum, Bacteroidete levels significantly decreased as the levels ofSI increased (a similar marginal effect was observed in ileum).Bacteroidete levels were lower among AUT-GI children compared toControl-GI children even after adjusting for the expression of alldisaccharidases and transporters.

TABLE 14 Multiple linear regression analysis examining disaccharidasesand transporters as predictors of bacterial levels among AUT-GI andControl-GI children. Interaction Outcome F-statistic Adjusted MainEffects: Coefficient Estimate Terms with Status Variable (p-value) R²Status SI^(STDev) MGAM^(STDev) LCT^(STDev) SGLT1^(STDev) GLUT2^(STDev)(Coefficient^(STDev)) Bacteroidetes, 5.52^(a) 0.56 −0.86*** −0.54† 0.05−0.02 0.35* 0.05 none Ileum—RT (0.003)** Bacteroidetes, 2.61^(a) 0.31−0.36* −0.60* 0.27 −0.08 0.29* 0.08 none Cecum—RT (0.062)† Firmicutes,2.50^(b) 0.33 0.40 −0.57† 0.44 −0.01 0.10 0.10 MGAM (−0.52)* Ileum—RT(0.068)† Firmicutes, 6.98^(c) 0.69 1.29*** −0.99** 0.86** 0.18† 0.060.40* MGAM (−0.50)*. Cecum—RT (0.001)** GLUT2 (−0.46)* Firm./Bac.,3.43^(b) 0.45 1.43** −0.19 0.19 0.04 −0.27 0.48† GLUT2 (−0.61)* Ileum—RT(0.024)* Firm./Bac., 5.13^(b) 0.58 1.47*** 0.27 0.21 0.19 −0.22 −0.02 SI(−0.93)** Cecum—RT (0.005)** Proteobacteria, 2.47^(b) 0.33 −1.05 2.76**−2.31* 0.01 −0.79† −0.59† MGAM (1.21)† Ileum—454 (0.071)†Proteobacteria, 5.41^(b) 0.59 −1.21 3.34*** −3.56*** −0.03 −0.68† −0.38MGAM (1.59)** Cecum—454 (0.004)** BetaProteobacteria, 1.14^(a) 0.04−0.14 0.61 −0.87 0.05 −0.26 −0.16 none Ileum—454 (0.385)BetaProteobacteria, 5.64^(a) 0.57 −0.16 1.43* −2.07** 0.27 −0.44 0.08none Cecum—454 (0.003)** ^(a)on 6 and 15 degrees of freedom ^(b)on 7 and14 degrees of freedom ^(c)on 8 and 13 degrees of freedom ^(STDev)Changein log-transformed outcome variable levels per unit standard deviationincrease in predictor variable (main effect variables or interactionterms) *p < 0.05; **p < 0.01; ***p < 0.001; †p < 0.1 (trend)

Finnicute levels significantly decreased as SI levels increased incecum. Cecal Firmicute levels were increased as the levels of MGAM andGLUT2 increased. The levels of Firmicutes in the cecum were higher inAUT-GI compared to Control-GI children after adjusting for theexpression of disaccharidases and transporters. Significant interactionwas found between status and MGAM and GLUT2 levels in the Firmicutemodels. Whereas higher levels of MGAM and GLUT2 were associated withhigher levels of Firmicutes among Control-GI children, the effects ofMGAM and GLUT2 on Firmicutes was not present in AUT-GI children.

Disaccharidases and transporter levels were not significant predictorsof the ratios of Firmicutes to Bacteroidetes in ileum or cecum. However,the interaction terms with GLUT2 in the ileum and SI in the cecum weresignificant.

Proteobacteria abundance significantly increased as the levels of SIincreased, but decreased as MGAM increased for both ileum and cecum.However, the interaction terms with MGAM in both ileum and cecum weresignificant, indicating that the magnitude of decline is significantlysmaller among AUT-GI children. Betaproteobacteria abundance waspositively associated with SI and inversely associated with MGAM only incecum; none of the interactions were significant. In addition,Proteobacteria and Betaproteobacteria abundance were not significantlydifferent between AUT-GI and Control-GI children after adjusting for theexpression of all disaccharidases and transporters. Overall, theseresults indicate that expression levels of disaccharidases andtransporters are associated with the abundance of Bacteroidetes,Firmicutes, and Betaproteobacteria in the mucoepithelium.

The levels of Betaproteobacteria in the ileum and cecum were higher inAUT-GI children with deficiencies in all 5 disaccharidases andtransporters versus AUT-GI children with fewer than 5 disaccharidase andtransporter deficiencies (FIG. 35A-B). Levels of CDX2 were lower inAUT-GI children with levels of Betaproteobacteria above the 75^(th)percentile of Control-GI children compared to AUT-GI children withlevels of Betaproteobacteria below the 75^(th) percentile of Control-GIchildren (FIG. 35C-D). These results indicate a potential link betweenincreased levels of Betaproteobacteria, reduced levels of CDX2expression, and overall deficiencies in disaccharidases andtransporters.

Timing of GI Disturbances Relative to Onset of Autism is Associated withChanges in Clostridiales Members

In this cohort, the onset of GI symptoms was reported to occur before orat the same time as the development of autism in 67% of AUT-GI children.As a sub-analysis, it was determined whether the timing of GI onsetrelative to autism onset was associated with gene expression andbacterial variables.

Patients were stratified based on whether the first episode of GIsymptoms occurred before or at the same time (within the same month) asthe onset of autism (AUT-GI-Before or Same group) or whether the firstepisode of GI symptoms occurred after the onset of autism (AUT-GI-Aftergroup). The timing of GI onset was not associated with levels ofdisaccharidase, hexose transporter or CDX2 transcripts, Bacteroidetes,Proteobacteria or Beta-proteobacteria (data not shown). However, asignificant effect was observed for the levels of Clostridiales andcumulative levels of Lachnospiraceae and Ruminococcaceae in both theileum and cecum (FIG. 31A-D). Whereas only a trend toward increasedClostridiales and cumulative levels of Lachnospiraceae andRuminococcaceae were observed when comparing all AUT-GI and Control-GIchildren (FIG. 27E-F and FIG. 18C-D), stratification based on timing ofGI onset revealed a significant increase in these variables in both theileum and cecum of the AUT-GI-Before or Same group relative to allControl-GI children (FIG. 31A: Clostridiales-ileum, Mann-Whitney,p=0.015; FIG. 31B: Clostridiales-cecum, Mann-Whitney, p=0.019; FIG. 31C:Lach.+Rum.-ileum, Mann-Whitney, p=0.015; FIG. 31D: Lach.+Rum.-cecum,Mann-Whitney, p=0.01 1). Furthermore, the levels of Clostridiales andcumulative levels of Lachnospiraceae and Ruminococcaceae weresignificantly higher in the AUT-GI-Before or Same group relative to theAUT-GI-After group (FIG. 31A: Clostridiales-ileum, Mann-Whitney,p=0.028; FIG. 31B: Clostridiales-cecum, Mann-Whitney, p=0.037; FIG. 31C:Lach.+Rum.-ileum, Mann-Whitney, p=0.028; FIG. 31D: Lach.+Rum.-cecum,Mann-Whitney, p=0.020); the AUT-GI-After group was not significantlydifferent from the Control-GI group. As expected, the AUT-GI-After grouphad a significantly older age at first onset of GI symptoms [median agein months, (interquartile range, IQR)=36, (22.5)] compared to theAUT-GI-Before or Same group [median age in months, (interquartile range,IQR)=1, (12)] (FIG. 31E: Mann-Whitney, p=0.007), and was also higherthan the Control-GI group [median age in months, (interquartile range,IQR)=1, (14)] (FIG. 31E: Mann-Whitney, p=0.027). The age at first GIonset was not significantly different between the AUT-GI-Before or Samegroup and the Control-GI group (FIG. 31E: Mann-Whitney, p=0.757). Thus,the increased levels of Clostridiales in the AUT-GI-Before or Same groupas compared to the Control-GI group were not influenced by differencesin age of onset of GI symptoms between these two groups. These resultsindicate that the timing of onset of GI symptoms relative to onset ofautism or the age at first GI onset can be associated with increases inClostridiales.

Associations Between Gene Expression, Bacterial Abundance, and FoodAllergies and Other Comorbid Atopic Manifestations

A National Survey of Children's Health performed under the auspices ofthe Centers for Disease Control reported that parents of autisticchildren reported more allergy symptoms than control children, and FAwere the most prevalent complaint [39]. Parental reports of FA in thecohort were reported with similar frequency in AUT-GI (67%) andControl-GI (71%) children. Milk-related (MA) and wheat-related (WA)allergies were the most commonly reported allergies in both groups(Table 6 and Table 7). To determine whether FA was associated with geneexpression or bacterial levels, patients in the AUT-GI group andControl-GI group were stratified based on reports of any FA (Table 15A),MA (Table 15B), or WA (Table 15C).

Stratification by any FA revealed a significant effect for levels ofGLUT2, ileal and cecal Firmicutes, ileal and cecal ratios of Firmicutesto Bacteroidetes, and cecal Betaproteobacteria (Table 15A). No effectwas observed for the levels of Bacteroidetes, which were significantlyreduced in AUT-GI children independent of FA status.

Stratification by MA status revealed even more significant effects(Table 15B). Significant effects were observed for MGAM, GLUT2, and CDX2expression, as well as ileal and cecal ratios of Firmicutes toBacteroidetes, and ileal and cecal Beta-proteobacteria. Additionaltrends were observed for SI expression and ileal and cecal Firmicutes.No effect was observed for the levels of Bacteroidetes, which weresignificantly reduced in AUT-GI children independent of MA status.

Stratification by WA status was associated with a significant effectonly for cecal levels of Firmicutes, though this effect was highlysignificant [AUT (+WA) vs. AUT (−WA): Mann-Whitney, p-value=0.008], andthe cecal ratio of Firmicutes to Bacteroidetes (Table 15C).

These results indicate that changes in the expression of somedisaccharidases and transporters and CDX2, as well as changes in theabundance of some bacterial phylotypes, are significantly associatedwith reported FA, especially MA. Whereas the levels of Firmicutes, theratio of Firmicutes to Bacteroidetes, and levels of Betaproteobacteriawere increased in AUT-GI children with FA, the levels of Bacteroideteswere not significantly different. This indicates that the levels ofBacteroidetes were significantly decreased in AUT-GI children,independent of FA status.

Atopic disease manifestations (AD: asthma, allergic rhinitis, or atopicdermatitis) were the most commonly reported comorbid conditions in bothAUT-GI and Control-GI children. The frequency of AD tended to be higherin the Control-GI group (100%) than in the AUT-GI group (53%) (Table 6:Fisher's Exact Test, 2-sided p=0.051). In the combined group (all AUT-GIand Control-GI patients), 86.7% of children with reported FA had atleast one reported AD; only 28.6% of children without reported foodallergy had one or more AD (Fisher's Exact Test, 2-sided p=0.014). As ADwas associated with reported FA, it was determined whether ADmanifestation was also associated with changes in disaccharidases andtransporters or bacterial parameters. Stratification of subjects by ADstatus revealed that cecal Firmicutes and the cecal ratio of Firmicutesto Bacteroidetes were higher in AUT-GI children with AD compared toControl-GI children with AD [Table 15D: AUT(+AD) vs. Control(+AD);Firmicutes CECRT, Mann-Whitney, p=0.015; Firm./Bacteroid. Ratio CECRT,Mann-Whitney, p=0.002] and AUT-GI children without AD [Table 15D:AUT(−AD) vs. AUT(+AD); Firmicutes CEC(RT), Mann-Whitney, p=0.049;Firm./Bacteroid. Ratio CEC(RT), Mann-Whitney, p=0.049].

TABLE 15A Association of food allergies (FA) with host gene expressionand bacterial phylotypes in AUT-GI children. AUT(+FA) vs. AUT(−FA) vs.Control(+FA)^(a), AUT(+FA)^(b), p-value^(MW), p-value^(MW), [effect in[effect in Variable AUT(+FA)] AUT(+FA)] GLUT2 0.014*, [decreased]0.037*, [decreased] Bacteroidetes IL(RT) 0.002**, [decreased] 0.806, [nochange] Bacteroidetes CEC(RT) 0.005**, [decreased] 0.713, [no change]Bacteroidetes IL(454) 0.037*, [decreased] 0.221, [no change]Bacteroidetes CEC(454) 0.050*, [decreased] 0.713, [no change] FirmicutesIL(RT) 0.221, [no change] 0.037*, [increased] Firmicutes CEC(RT) 0.037*,[increased] 0.010*, [increased] Firm./Bacteroid. Ratio 0.003**,[increased] 0.037*, [increased] IL(RT) Firm./Bacteroid. Ratio 0.005**,[increased] 0.020*, [increased] CEC(RT) Beta-proteobacteria IL(454)0.050†, [increased] 0.066†, [increased] Beta-proteobacteria 0.028*,[increased] 0.037*, [increased] CEC(454) ^(a)AUT(+FA), n = 10;Control(+FA), n = 5 ^(b)AUT(−FA), n = 5; AUT(+FA), n = 10^(MW)Mann-Whitney test

TABLE 15B Association of milk allergies (MA) with host gene expressionand bacterial phylotypes in AUT-GI children. AUT(+MA) vs. AUT(−MA) vs.Control(+MA)^(a), AUT(+MA)^(b), p-value^(MW), p-value^(MW), [effect in[effect in Variable AUT(+MA)] AUT(+MA)] SI 0.006**, [decreased] 0.099†,[decreased] MGAM 0.006**, [decreased] 0.045*, [decreased] GLUT2 0.009**,[decreased] 0.013*, [decreased] CDX2 0.072†, [decreased] 0.034*,[decreased] Bacteroidetes IL(RT) 0.003**, [decreased] 0.480, [no change]Bacteroidetes CEC(RT) 0.003**, [decreased] 0.289, [no change]Bacteroidetes IL(454) 0.028*, [decreased] 0.637, [no change]Bacteroidetes CEC(454) 0.020*, [decreased] 0.637, [no change] FirmicutesIL(RT) 0.205, [no change] 0.059†, [increased] Firmicutes CEC(RT) 0.053†,[increased] 0.099†, [increased] Firm./Bacteroid. Ratio 0.004**,[increased] 0.034*, [increased] IL(RT) Firm./Bacteroid. Ratio 0.006**,[increased] 0.045*, [increased] CEC(RT) Beta-proteobacteria 0.020*,[increased] 0.013*, [increased] IL(454) Beta-proteobacteria 0.009**,[increased] 0.007**, [increased] CEC(454) ^(a)AUT(+MA), n = 9;Control(+MA), n = 5 ^(b)AUT(−MA), n = 6; AUT(+MA), n = 9^(MW)Mann-Whitney test

TABLE 15C Association of wheat allergies (WA) with host gene expressionand bacterial phylotypes in AUT-GI children. AUT(+WA) vs. AUT(−WA) vs.Control(+WA)^(a), AUT(+WA)^(b), p-value^(MW), p-value^(MW), Variable[effect in AUT(+WA)] [effect in AUT(+WA)] Bacteroidetes IL(RT) 0.007**,[decreased] 0.643, [no change] Bacteroidetes CEC(RT) 0.017*, [decreased]0.643, [no change] Bacteroidetes IL(454) 0.017*, [decreased] 0.488, [nochange] Bacteroidetes CEC(454) 0.089†, [decreased] 0.908, [no change]Firmicutes IL(RT) 0.174, [no change] 0.083†, [increased] FirmicutesCEC(RT) 0.089†, [increased] 0.008*, [increased] Firm./Bacteroid. Ratio0.011*, [increased] 0.203, [no change] IL(RT) Firm./Bacteroid. Ratio0.011*, [increased] 0.049*, [increased] CEC(RT) Beta-proteobacteria0.089†, [increased] 0.643, [no change] IL(454) Beta-proteobacteria0.042*, [increased] 0.418, [no change] CEC(454) ^(a)AUT(+WA), n = 8;Control(+WA), n = 4 ^(b)AUT(−WA), n = 7; AUT(+WA), n = 8^(MW)Mann-Whitney test

TABLE 15D Association of atopic disease (AD) status with host geneexpression and bacterial phylotypes in AUT-GI children. AUT(+AD) vs.AUT(−AD) vs. Control(+AD)^(a), AUT(+AD)^(b), p-value^(MW), p-value^(MW),Variable [effect in AUT(+AD)] [effect in AUT(+AD)] Bacteroidetes IL(RT)0.008**, [decreased] 0.563, [no change] Bacteroidetes CEC(RT) 0.028*,[decreased] 0.418, [no change] Bacteroidetes IL(454) 0.049*, [decreased]0.643, [no change] Bacteroidetes CEC(454) 0.064†, [decreased] 0.908, [nochange] Firmicutes IL(RT) 0.064†, [increased] 0.133, [no change]Firmicutes CEC(RT) 0.015*, [increased] 0.049*, [increased]Firm./Bacteroid. Ratio 0.002**, [increased] 0.064†, [increased] IL(RT)Firm./Bacteroid. Ratio 0.006**, [increased] 0.049*, [increased] CEC(RT)Beta-proteobacteria 0.049*, [increased] 0.203, [no change] IL(454)Beta-proteobacteria 0.028*, [increased] 0.133, [no change] CEC(454)^(a)AUT(+AD), n = 8; Control(+AD), n = 7 ^(b)AUT(−AD), n = 7; AUT(+AD),n = 8 ^(MW)Mann-Whitney test

Discussion

Although the major deficits in ASD are social and cognitive, manyaffected individuals with ASD also have substantial GI morbidity.Findings in this study that can shed light on GI morbidity in ASDinclude the observations that: (1) levels of transcripts fordisaccharidases and hexose transporters are reduced in AUT-GI children;(2) AUT-GI children have microbial dysbiosis in the mucoepithelium; and(3) dysbiosis is associated with deficiencies in host disacharidase andhexose transporter mRNA expression. Without being bound by theory,deficiencies in disaccharidases and hexose transporters alter the milieuof carbohydrates in the distal small intestine (ileum) and proximallarge intestine (cecum), resulting in the supply of additional growthsubstrates for bacteria. These changes manifest in significant andspecific compositional changes in the microbiota of AUT-GI children (seeFIG. 32, FIG. 20).

A previous report on GI disturbances in ASD found low activities of atleast one disaccharidase or glucoamylase in duodenum in 58% of children[7]. In this study, 93.3% of AUT-GI children had decreased mRNA levelsfor at least one of the three disaccharidases (SI, MGAM, or LCT). Inaddition, decreased levels of mRNA were found for two important hexosetransporters, SGLT1 and GLUT2. Congenital defects in these enzymes andtransporters are extremely rare [40,41], and even the common variant foradult-type hypolactasia was not responsible for reduced LCT expressionin AUT-GI children in this cohort. It is unlikely, therefore, that thecombined deficiency of disaccharidases (maldigestion) and transporters(malabsorption) are indicative of a primary malabsorption resulting frommultiple congenital or acquired defects in each of these genes:Transcripts for the enterocyte marker, villin, were not reduced inAUT-GI ilea and did not predict the expression levels of any of thedisaccharidases or transporters in multiple regression models. Thisindicates that a general loss of enterocytes is unlikely. Without beingbound by theory, defects in the maturational status of enterocytes orenterocyte migration along crypt-villus axis can contribute to deficitsin disaccharidase and transporter expression [42].

The ileal expression of CDX2, a master transcriptional regulator in theintestine, was a significant predictor of mRNA expression of all fivedisaccharidases and transporters in AUT-GI and Control-GI children basedon linear regression models. However, as ASD status remained asignificant predictor of disaccharidase mRNA expression even afteradjusting for CDX2 and villin, additional factors must also contribute.One factor is diet. Dietary intake of carbohydrates can regulate themRNA expression of disaccharidases and hexose transporters in mice andrats [43,44,45]. ASD children exhibit feeding selectivity and aberrantnutrient consumption [46,47,48,49,50,51,52]. However, of the fourstudies reporting carbohydrate intake, none found differences in totalcarbohydrate intake in ASD children [47,48,49,50]. Furthermore, onestudy found no association between dietary intake of macronutrients(i.e., carbohydrates, proteins, or fats) and GI symptoms [47].Unfortunately, dietary diaries for the period immediately precedingbiopsy were not available for the children evaluated in this study;hence, the extent to which dietary intake affected intestinal geneexpression could not be determined.

Hormonal and growth factor regulation of some disaccharidases and hexosetransporters have been reported in in vitro studies and in animals[53,54]. Inflammatory cytokines can regulate SI gene expression in humanintestinal epithelial cells in vitro [55]. Thus, immunological orhormonal imbalances reported in ASD children [5,8,9,10,11,12,56,57,58]can also contribute to expression deficits. Additionally, intestinalmicrobes can influence the expression of disaccharidases andtransporters [59] through the influence of pathogen-associated molecularpatterns (PAMPs) and butyrate (a byproduct of bacterial fermentation) onCDX2 expression and activity [60,61,62,63]. In this regard, theobservation that CDX2 was decreased in AUT-GI children with increasedlevels of Betaproteobacteria can be important.

Whatever the underlying mechanisms, reduced capacity for digestion andtransport of carbohydrates can have profound effects. Within theintestine, malabsorbed carbohydrates can lead to osmotic diarrhea [64];non-absorbed sugars can also serve as substrates for intestinalmicroflora that produce fatty acids and gases (methane, hydrogen, andcarbon dioxide), promoting additional GI symptoms such as bloating andflatulence [65]. The deficiency of even a single gene in this importantpathway can result in severe GI disease, as occurs withglucose-galactose malabsorption syndrome caused by SGLT1 deficiency,Fanconi-Bickel syndrome resulting from GLUT2 mutations,sucrase-isomaltase deficiency, and congenital lactase deficiency[40,41].

Changes in the type and quantity of dietary carbohydrates can influencecomposition and function of intestinal microflora [66,67,68]; thus, wereasoned that carbohydrate maldigestion and malabsorption, resultingfrom deficient expression of disaccharidases and hexose transporters,might have similar effects in AUT-GI subjects. Pyrosequencing analysisof mucoepithelial bacteria revealed significant multicomponent dysbiosisin AUT-GI children, including decreased levels of Bacteroidetes, anincrease in the Firmicute/Bacteroidete ratio, increased cumulativelevels of Firmicutes and Proteobacteria, and an increase in levels ofbacteria in the class Betaproteobacteria.

A recent pyrosequencing study reported an increase in Bacteroidetes infecal samples of ASD subjects [69]. Although these findings can appearto be incongruent with those reported here, the data were obtained usingbiopsies rather than free fecal material. Others have reporteddifferences in the composition of fecal versus mucosal microflora[35,70,71,72]. Only about 50% of cells in feces are viable, with deadand injured cells making up the remaining fractions [73]. The loss ofBacteroidetes from the mucoepithelium as a result of death, injury, orcompetition for binding in the mucosal space can result in increasedwash out of Bacteroidete cells into the fecal stream. Thus, higherlevels of Bacteroidetes in feces could be indicative of an inability tothrive in the mucosal microbiome rather than an indication thatBacteroidetes are found at higher levels in the microbiome.

The trend toward increased Firmicutes was largely attributable toClostridia with Ruminococcaceae and Lachnospiraceae as majorcontributors. Several Ruminococcaceae and Lachnospiraceae are knownbutyrate producers and can thus influence short-chain fatty acid (SCFA)levels [74]. SCFA influence colonic pH, and some Bacteroides sp. aresensitive to acidic pH [75]. Three previous reports indicateddifferences in Clostridia species in children with ASD, includinggreater abundance of Clostridium clusters I, II, XI and C. bolteae[14,15,16]. Stratification of AUT-GI children based on the timing of GIsymptom development relative to autism onset revealed that the levels ofClostridiales and cumulative levels of Lachnospiraceae andRuminococcaceae were significantly higher in AUT-GI children for whom GIsymptoms developed before or at the same time as the onset of autismsymptoms compared to AUT-GI children for whom GI symptoms developedafter the onset of autism and compared to Control-GI children. However,we cannot discern whether changes in Clonstridiales members occurredbefore the onset of autism in this subgroup. We can only conclude thatincreased levels of Clostridiales members in biopsies taken after thedevelopment of both GI symptoms and autism are associated with thetiming of GI onset relative to autism onset in this cohort. Although thereason for this association remains unclear, this finding can indicatethat the timing of GI onset relative to autism is an important variableto consider in the design of future prospective studies investigatingthe microbiota of children with autism.

Although we found only a trend for increased Firmicutes in AUT-GIchildren, the cumulative levels of Firmicutes and Proteobacteria weresignificantly higher. These results indicate that in some patients thedecrease in Bacteroidetes is associated with an increase in Firmicutes,whereas in others increases in Proteobacteria are associated with areduced abundance of Bacteroidetes. Three AUT-GI patients had highlevels of Alpha-, Beta-, or Gammaproteobacteria. In addition, the AUT-GIgroup had elevated levels of Betaproteobacteria compared to theControl-GI group, primarily reflecting the presence of Alcaligenaceae.Alcaligenaceae sequences were not detected in any tissues fromControl-GI children.

Deficient digestion and absorption of di- and monosaccharides in thesmall intestine can alter the balance of growth substrates, eliminatingthe growth advantages that Bacteroidetes enjoy in the healthy intestineand enabling competitive growth of bacterial phylotypes better suitedfor growth on undigested and unabsorbed carbohydrates. In support ofthis hypothesis, multiple linear regression models demonstrated thatlevels of ileal SGLT1 and Si mRNA were associated with levels ofBacteroidetes in ileum and cecum, or cecum alone, respectively. Levelsof ileal SI, MGAM and GLUT2 mRNA were associated with levels of cecalFirmicutes, although the magnitude of the effects of MGAM and GLUT2differed between AUT-GI and Control-GI children. Significantassociations were also observed between levels of SI and MGAM mRNA andof Proteobacteria in ileum and cecum, and of Betaproteobacteria incecum. Although deficiencies in disaccharidase and transporterexpression appear to at least partially contribute to these alterationsin the AUT-GI microbiota, diagnostic status remained a significantpredictor of Bacteroidete and cecal Firmicute abundance even afteradjusting for gene expression.

Metabolic interactions between intestinal microflora and their hosts areonly beginning to be understood. Nonetheless, there is already abundantevidence that microflora can have system-wide effects[76,77,78,79,80,81,82,83] and influence immune responses, braindevelopment and behavior [24,25,26,84,85]. We acknowledge that this is asmall study comprising 22 subjects. The small sample size evaluated inthis study is a limitation arising from the difficulty in obtainingbiopsies from young children undergoing invasive endoscopic examination.Without being bound by theory, the data show that at least some childrenwith autism have a distinct intestinal profile that is linked todeficient expression of disaccharidases and hexose transporters,potentially promoting maldigestion, malabsorption and multicomponent,compositional dysbiosis. Although the underlying cause of these changesand the extra-intestinal effects these changes can elicit remainspeculative, the identification of specific molecular and microbialsignatures that define GI pathophysiology in AUT-GI children sets thestage for further research aimed at defining the epidemiology, diagnosisand informed treatment of GI symptoms in autism.

Materials and Methods

All samples were analyzed anonymously. Samples assessed in this examplewere restricted to those derived from male children from the originalcohort between 3 and 5 years of age to control for confounding effectsof gender and age on intestinal gene expression and the microbiota. Thissubset comprised 15 AUT-GI (Patient #1-15) and 7 Control-GI (Patient#16-22) patients.

Clinical Procedures: Specific clinical procedures for definingneuropsychiatric and regression status in this cohort have beenpreviously described [86]. Briefly, neuropsychiatric status wasestablished for all subjects using Diagnostic and StatisticalManual-Fourth Edition, Text Revision (DSM-IV-TR) diagnostic criteria.Only cases meeting full DSM-IV-TR criteria for Autistic Disorder (AUT)were included for further analysis. DSM-IV-TR diagnosis of AUT wasconfirmed by certified raters using the Autism DiagnosticInterview-Revised (ADI-R). Regression status was determined based onADI-R and Shortened CPEA Regression Interview. Control-GI children wereevaluated in the same manner as cases to exclude subjects with anydevelopmental disturbances, including ASD. Age of AUT onset wasdetermined by an ADI-R certified interviewer. Questions posed to parentsin standardized data collection forms regarding GI symptoms were basedon previous work [27]. Symptoms were only reported if the child hadexperienced the specific GI symptoms, including food allergies, for 3consecutive months. History of medication use, presence of comorbidconditions, age at first GI episode, and presence and type of foodallergies were also acquired through parental questionnaires.

RNA and DNA extraction: All biopsies were snap frozen at collection andstored at -80° C. until extraction. RNA and DNA were extractedsequentially from individual ileal and cecal biopsies [total of 176biopsies from 15 AUT-GI patients and 7 Control-GI patients: 8 biopsiesper patient (4 each from ileum and cecum), yielding 88 ileal and 88cecal biopsies] in TRIzol (Invitrogen) using standard protocols. RNAfrom half of the biopsies (2 ileal and 2 cecal biopsies per AUT-GI orControl-GI patient) was derived from residual extracts from the originalstudy completed in 2008 [86]. RNA from the other half of the biopsies(the remaining 2 ileal and 2 cecal biopsies per AUT-GI or Control-GIpatient) was newly extracted from stored biopsies (stored undisturbed at−80° C.) at the inception of the current study in 2008. The interphaseand organic phase fractions were stored at −80° C., following RNAextraction, for subsequent DNA extraction. All extractions were storedin aliquots at −80° C. to avoid repeated freeze thawing of samples. RNAand DNA concentrations, purity and integrity were determined for allresidual extracts and newly extracted biopsies just prior to cDNAsynthesis for mRNA expression studies and just prior to PCR of newlyextracted DNA using a Nanodrop ND-1000 Spectrophotometer (NanodropTechnologies) and Bioanalyzer (Agilent Technologies).

Quantitative Real-Time PCR of human mRNA: Intron/exon spanning,gene-specific PCR primers and probes (Table 16) for SI, MGAM, LCT,SGLT1, GLUT2, villin, and CDX2, with GAPDH and β-actin as dualhousekeeping gene controls were designed for real-time PCR using PrimerExpress 1.0 software (Applied Biosystems). Taqman probes were labeledwith the reporter FAM (6-carboxyfluorescein) and the quencher BBQ(Blackberry) (TIB MolBiol). Assays were designed and implemented aspreviously described [87,88,89]. Levels of mRNA expression for each geneand in each AUT-GI individual were considered significantly increased ordecreased if they were above the 75^(th) percentile or below the 25^(th)percentile, respectively, of gene expression obtained for all Control-GIchildren and were at least 2-fold increased or decreased from theControl-GI mean (Table 9 and Table 10).

TABLE 16 Real-time PCR primers and probes used for gene expression andbacterial quantitative analysis. Amplicion Name SEQ ID Primers and Probesize (bp) SI 26 For: 5′-TCTTCATGAGTTTTATGAGGATACGAAC-3′  150 27 Rev:5′-TTTGCACCAGATTCATAATCATACC-3′ 27 Probe:5′-CAGATACTGTGAGTGCCTACATCCCTGATGCTATT-3′ MGAM 29 For:5′-TACCTTGATGCATAAGGCCCA-3′  150 30 Rev: 5′-GGCATTACGCTCCAGGACA-3′ 31Probe: 5′-CGTCACTGTTGTGCGGCCTCTGC-3′ LCT 32 For:5′-CAGGAATCAAGAGCGTCACAACT-3′  180 33 Rev: 5′-AAATCGACCGTGTCCTGGG-3′ 34Probe: 5′-TCCTGCTAGAACCACCCATATCTGCGCT-3′ SGLT1 35 For:5′-GCTCATGCCCAATGGACTG-3′  125 36 Rev: 5′-CGGACCTTGGCGTAGATGTC-3′ 37Probe: 5′-ACAGCGCCAGCACCCTCTTCACC-3′ Glut2 38 For:5′-AGTTAGATGAGGAAGTCAAAGCAA-3′  164 39 Rev: 5′-TAGGCTGTCGGTAGCTGG-3′ 40Probe: 5′-ACAAAGCTTGAAAAGACTCAGAGGATATGATGATGTC-3′ Villin 41 For:5′-CATGCGCTGAACTTCATCAAA-3′  120 42 Rev: 5′-GGTTGGACGCTGTCCACTTC-3′ 43Probe: 5′-CGGCCGTCTTTCAGCAGCTCTTCC-3′ CDX2 44 For:5′-GGCAGCCAAGTGAAAACCAG-3′  112 45 Rev: 5′-TCCGGATGGTGATGTAGCG-3′ 46Probe: 5′-ACCACCAGCGGCTGGAGCTGG-3′ β-Actin 47 For:5′-AGCCTCGCCTTTGCCGA-3′  175 48 Rev: 5′-CTGGTGCCTGGGGCG-3′ 49 Probe:5′-CCGCCGCCCGTCCACACCCGCC GAPDH 50 For: 5′-CCTGTTCGACAGTCAGCCG-3′  10051 Rev: 5′-CGACCAAATCCGTTGACTCC-3′ 52 Probe: 5′-CGTCGCCAGCCGAGCCACA-3′Bacteroidetes 53 For: 5′-AACGCTAGCTACAGGCTT-3′ ~293 54 Rev:5′-CCAATGTGGGGGACCTTC-3′ Firmicules 55 For:5′-GGAGYATGTGGTTTAATTCGAAGCA-3′ ~126 56 Rev: 5′-AGCTGACGACAACCATGCAC-3′Total Bacteria 57 For: 5′-GTGCCAGCMGCCGCGGTAA-3′ ~295 58 Rev:5′-GACTACCAGGGTATCTAAT-3′

Lactase genotyping: Genomic DNA from AUT-GI and Control-GI patients wassubjected to previously-described, PCR-restriction fragment lengthpolymorphism (PCR-RFLP) analysis for the C/T-13910 and G/A-22018polymorphisms associated with adult-type hypolactasia with minormodifications [90]. For details, see FIG. 21B-E.

Barcoded pyrosequencing of intestinal microbiota: PCR was carried outusing bacterial 16S rRNA gene-specific (V2-region), barcoded primers aspreviously described [91]. Barcoded 16S rRNA genes were amplified fromDNA samples from the 88 ileal biopsies and 88 cecal biopsies. Ampliconswere sequenced at 454 Life Sciences on a GS FLX sequencer.

Quantitative Real-time PCR of Bacteroidete and Firmicute 16S rRNA genes:Primer sequences and PCR conditions used for bacterial real-time PCRassays to quantify Bacteroidetes, Firmicutes, and total Bacterial 16SrRNA genes have been previously described [92,93]; primer sequences arelisted in Table 16. Copy numbers of Bacteroidetes, Firmicutes, orFirmicute to Bacteroidete ratios that were above the 75^(th) percentileor below the 25^(th) percentile of Control-GI children were scored as anincrease or decrease, respectively (Table 9). Percent changes inbacterial parameters for individuals in the AUT-GI group were determinedbased on the mean levels in Control-GI children (Table 12).

Bioinformatic analysis of pyrosequencing reads: Pyrosequencing readsranging from 235 to 300 base pairs in length (encompassing all sequenceswithin the major peak obtained from pyrosequencing) were filtered foranalysis. Low-quality sequences—i.e., those with average quality scoresbelow 25—were removed based on previously described criteria [91,94].Additionally, reads with any ambiguous characters were omitted fromanalysis. Sequences were then binned according to barcode, followed byremoval of primer and barcode sequences. Taxonomic classifications ofbacterial 16S rRNA sequences were obtained using the RDP classifier toolwith a minimum 80% bootstrap confidence estimate. To normalize data fordifferences in total sequences obtained per patient, phylotype abundancewas expressed as a percentage of total bacterial sequence reads perpatient at all taxonomic levels. Taxonomy note: the RDP classifierbinned all of the limited number of sequences obtained for the phylumCyanobacteria into the chloroplast-derived genus Streptophyta. Heatmapswere constructed with MeV (Version 4.5.0), using abundance data frompyrosequencing reads. Heatmap scales were made linear where possible,with the upper limit reflecting the highest abundance recorded for anytaxa in a given heatmap (red), the lower limit reflecting sequencesabove 0% abundance (green), and the midpoint limit (white) set to thetrue midpoint between 0% and the upper limit. In some instances, themidpoint limit was adjusted to highlight salient differences between theAUT-GI and Control-GI groups. Gray cells in all heatmaps reflect thecomplete absence of sequences detected for a given taxa in a givenpatient.

OTU-based analysis was carried out in MOTHUR (version 1.8.0) [95].Filtered sequences generated from 454 pyrosequencing were aligned to thegreengenes reference alignment (greengenes.lbl.gov), using theNeedleman-Wunsch algorithm with the “align.seqs” function (ksize=9).Pairwise genetic distances among the aligned sequences were calculatedusing the “dist.seqs” function (calc=onegap, countends=T). Sequenceswere assigned to OTUs (97% identity) using nearest neighbor clustering.Rarefaction curves to assess coverage and diversity (Shannon DiversityIndex) were constructed in MOTHUR. For OTU analysis of Bacteroidetesequences, phylum level classification in RDP was used to subselect allBacteroidete sequences, followed by OTU assignment at 97% identity.Representative sequences (defined as the sequence with the minimumdistance to all other sequences in the OTU) from each OTU were obtainedusing the get.oturep command in MOTHUR. Representative sequences wereclassified using the nearest species match from Greengenes Blast(greengenes.lbl.gov) and NCBI BLAST alignment. OTU abundance by patientwas expressed as percent relative abundance, determined by dividing thenumber of reads for an OTU in a given patient sample by the total numberof bacterial reads obtained through pyrosequencing for that sample.

Statistical analysis: Most of the data were not normally distributed,based on Kolmogorov-Smirnov test and evaluation of skewness andkurtosis; thus, the non-parametric Mann-Whitney U test was performed toevaluate differences between groups using StatView (Windows version5.0.1; SAS Institute). The comparative results of gene expression andbacteria 16S rRNA gene levels were visualized as box-and-whisker plotsshowing: the median and the interquartile (midspread) range (boxescontaining 50% of all values), the whiskers (representing the 25^(th)and 75^(th) percentiles) and the extreme data points (open circles).Chi-squared test was used to evaluate between-group genotypes foradult-type hypolactasia as well as differences in the frequency ofatopic disease between groups. Kruskal-Wallis one-way analysis ofvariance was employed to assess significance of LCT mRNA expressionlevels split by genotype and group. To evaluate the effects of CDX2and/or villin on enzyme and transporter levels and the effects of levelsof enzymes and transporters on bacterial levels, multiple linearregression analyses were conducted. For details on multiple linearregression analyses see Table 11, and Table 14. Significance wasaccepted for all analyses at p<0.05.

Supporting Results

Genetically determined lactase non-persistence is not responsible fordeficient lactase mRNA in AUT-GI children (FIG. 21): Although it isbeyond the scope of this study to evaluate all possible mutations incarbohydrate genes that can affect expression, it was confirmed thatdeficient LCT mRNA in AUT-GI children is not a result of the commonadult-type hypolactasia genotype. LCT mRNA levels can be affected by twosingle nucleotide polymorphisms that determine adult-type hypolactasia;therefore, these children were genotyped using PCR-RFLP analysis. Thehomozygous, hypolactasia variant alleles were found in 20% (3 out of 15)of AUT-GI children and 14.3% (1 out of 7) of Control-GI children.Genotype proportions were not significantly different between the twogroups (chi-squared test, p=0.896) (FIG. 21B). LCT mRNA expression wassignificantly lower in individuals with the homozygous hypolactasiagenotype compared to all other genotypes (FIG. 21C: Mann-Whitney,p=0.033). Comparison of LCT mRNA expression across genotype and groupfailed to reach significance (FIG. 21D: Kruskal-Wallis, p=0.097).Comparison of mRNA expression in subjects carrying at least one copy ofthe normal allele confirmed a significant decrease in LCT mRNA in AUT-GIrelative to Control-GI subjects, independent of the individuals with thehomozygous hypolactasia genotype (FIG. 21E: Mann-Whitney, p=0.025). Insummary, although the data support the notion that LCT genotype affectsgene expression, deficient LCT mRNA in AUT-GI was not attributable todisproportionate hypolactasia genotypes between the AUT-GI andControl-GI groups.

Barcoded 16S rRNA gene pyrosequencing (FIG. 23): A total of 525,519sequencing reads (representing 85% of the initial number of sequencingreads) remained after filtering based on read length, removinglow-quality sequences and combining duplicate pyrosequencing runs(271,043 reads for ilea; 254,476 reads for ceca). Binning of sequencesby barcode revealed similar numbers of 16S rRNA gene sequence reads perpatient (average # sequences per patient+/−STD for ilea=12,320+/−1220;average # sequences per patient+/−STD for ceca=11,567+/−1589). There wasnot a significant difference between the AUT-GI and Control-GI groups interms of the number of reads per patient. In order to assess whethersufficient sampling was achieved in the total pyrosequencing data setfor all AUT-GI and Control-GI subjects, OTUs (Operational TaxonomicUnits) were defined at a threshold of 97% identity, split by data forileum and cecum, and rarefaction analysis was carried out (FIG. 23A-B).Rarefaction curves showed a tendency toward reaching plateau for allsubjects; however failure to reach plateau indicates that additionalsampling would be required to achieve complete coverage of all OTUspresent in ileal and cecal biopsies. Investigation of diversity inAUT-GI and Control-GI patients was carried out using the ShannonDiversity Index calculated from OTU data for each subject. Rarefactionanalysis revealed that all Shannon Diversity estimates had reachedstable values (FIG. 23C-D). While Shannon Diversity estimates variedwidely between individuals, there was not an apparent overall difference(loss or gain of diversity) between the AUT-GI and Control-GI groups inileal (FIG. 23C) or cecal (FIG. 23D) biopsies.

OTU Analysis of Bacteroidetes (FIG. 25): In order to determine whetherthe decreased abundance of Bacteroidete members was attributable to theloss of specific Bacteroidete phylotypes, the distribution ofBacteroidete OTUs (defined using a threshold of 97% identity or greater;3% distance) were investigated. The number of Bacteroidete OTUs perpatient ranged from 23 to 102 for ileal samples and 10 to 130 for cecalsamples. Interestingly, no single OTU was significantly over orunderrepresented between AUT-GI and Control-GI children and many OTUscontained single sequences. Furthermore, high inter-subject variabilityin the distribution and abundance of individual Bacteroidete phylotypeswas observed, as has been previously described [B1]. Thus, it wasdetermined whether the decrease in Bacteroidete abundance in AUT-GIchildren could be attributed to overall losses of the most prevalentBacteroidete phylotypes. In both ileal and cecal samples, 12 OTUsaccounted for the majority of Bacteroidete sequences (FIG. 25A-B). Thecumulative levels of these 12 OTUs were significantly lower in AUT-GIcompared to Control-GI children in both the ileum (FIG. 25C:Mann-Whitney, p=0.008) and cecum (FIG. 25D: Mann-Whitney, p=0.008).Representative sequences from each of these 12 OTUs were classifiedusing Greengenes Blast and microbial blast alignment (NCBI) (FIG. 25E).The majority of sequences were members of the family Bacteroidaceae(OTUs 1, 3, 5, 6, 7, and 19), except in the case of patient 20, wherePrevotellaceae were the dominant phylotype (OTU #21). These resultsindicate that the loss of Bacteroidetes in AUT-GI children is primarilyattributable to overall decreases in the dominant phylotypes ofBacteroidetes in individual patients

Evaluation of confounding effects of probiotic, proton-pump inhibitorand antibiotic use: The use of probiotics (Pb), proton-pump inhibitors(PPI), and antibiotics are reported to exert effects on the compositionof the intestinal microbiota [B2, B3]. As some patients in both theAUT-GI and Control-GI groups had taken these medications, we sought todetermine whether potential confounding effects of these medications onthe findings could be excluded. Probiotics had been used by both AUT-GI(n=4; 27%) and Control-GI (n=1; 14%) children. If probiotics usedetermined the outcome of gene expression and bacterial variables, thenthe significant effect for a given variable should not be present whencomparing individuals that had not taken probiotics in the AUT-GI andControl-GI groups [Table 13A: AUT(−Pb) vs. Control(−Pb)]. For each ofthe 16 variables, except the ratio of Firmicutes to Bacteroidetes in thececum (RT) and Betaproteobacteria in the cecum (454), either asignificant result or trend was observed between the AUT(-Pb) andControl(-Pb) groups. If the cecal ratio of Firmicutes to Bacteroidetesand Betaproteobacteria are affected by probiotic use, then a differencein the levels of these bacterial parameters should be evident whencomparing AUT-GI probiotic non-users vs. AUT-GI probiotic users [TableSSA: AUT(−Pb) vs. AUT(+Pb)]. There was not a significant difference inBetaproteobacteria levels between these groups; however, the ratio ofFirmicutes to Bacteroidetes in the cecum, determined by real-time PCR,was significantly higher in the AUT(+Pb) group compared to the AUT(−Pb)group (Table 13A: Mann-Whitney, p=0.037). Thus an effect mediated byprobiotics on this variable cannot be excluded. This effect, however,was not apparent in the corresponding ratio of Firmicutes toBacteroidetes in the cecum, determined by pyrosequencing.

The use of proton-pump inhibitors (PPI: Lanzoprazole or Omeprazole) wassimilarly examined. PPI had been used by both AUT-GI (n=4; 27%) andControl-GI (n=2; 29%) children. A significant difference was found forall variables, except LCT, when comparing AUT(−PPI) children withControl(−PPI) children [Table SSB: AUT(−PPI) vs. Control(−PPI)]. Thus apotential effect of PPI use should only be considered for LCT. As LCTlevels were not significantly different between AUT(−PPI) and AUT(+PPI)children, it is unlikely that PPIs exerted any major effect on LCTexpression. A trend toward an effect in the levels of Bacteroidetes inthe ileum, determined by pyrosequencing, was evident between AUT(−PPI)and AUT(+PPI) children; however, a significant effect was observedbetween AUT(−PPI) and Control(−PPI) children. This indicates that thispotential effect was not a major determinant of the difference in ilealBacteroidetes between AUT-GI and Control-GI children. Only one patient(AUT-GI patient #1) had used both probiotics and proton-pump inhibitors,thus an additive effect was not evaluated. Grouping of patients based onwhether they had taken either probiotics or proton-pump inhibitors didnot reveal any significant effects in the 16 variables.

Only one individual had taken an antibiotic (amoxicillin) in this cohort(Control-GI patient #16). This patient had high levels of mRNAexpression for all disaccharidases and transporters, within the range ofother Control-GI children and at least above the 90^(th) percentile ofall AUT-GI children. Thus, exclusion of this patient from the analysishad a negative effect on significance values obtained for geneexpression assays (Table 13C). These results indicate that antibioticuse had no effect on disaccharidase and hexose transporter levels inthis patient. In contrast, Control-GI patient #16 consistently had thelowest levels of Bacteroidetes (representing the low-range outlier)compared to all other Control-GI children in pyrosequencing andreal-time PCR assays. Thus, exclusion of this patient from analysis ofbacterial phylotypes generally improved the significance of resultsobtained for Bacteroidetes, ratios of Firmicutes to Bacteroidetes, andratios of Clostridiales to Bacteroidales. Levels of Beta-proteobacteriain the cecum for this patient were near the median value of all otherControl-GI children. Thus, it is likely that antibiotic use in thispatient had some effect on Bacteroidete levels, but no effect onBetaproteobacteria or gene expression for disaccharidases andtransporters. As the effect of antibiotic use in this patient did notaffect all variables and exclusion of this patient did not affect theinterpretation of results, this patient was not excluded from theoverall analysis.

REFERENCES FOR SUPPORTING RESULTS

-   B1. Eckburg P B, Bik E M, Bernstein C N, Purdom E, Dethlefsen L, et    al. (2005) Diversity of the human intestinal microbial flora.    Science 308: 1635-1638.-   B2. Reid G, Younes J A, Van der Mei H C, Gloor G B, Knight R, et    al. (2011) Microbiota restoration: natural and supplemented recovery    of human microbial communities. Nat Rev Microbiol 9: 27-38.-   B3. Lombardo L, Foti M, Ruggia O, Chiecchio A (2010) Increased    incidence of small intestinal bacterial overgrowth during proton    pump inhibitor therapy. Clin Gastroenterol Hepatol 8: 504-508.

Supporting Methods

Quantitative Real-Time PCR of human mRNA: PCR standards for determiningcopy numbers of target transcripts were generated from amplicons of SI,MGAM, LCT, SGLT1, GLUT2, Villin, CDX2, GAPDH, and Beta-actin cloned intothe vector pGEM-T easy (Promega Corporation). Linearized plasmids werequantitated using a Nanodrop ND-1000 Spectrophotometer, and 10-foldserial dilutions (ranging from 5×10⁵ to 5×10⁰ copies) were created inwater containing yeast tRNA (1 ng/μl). Unpooled RNA from individualileal biopsies were used for real time PCR assays; each individualbiopsy was assayed in duplicate. cDNA was synthesized using Taqmanreverse transcription reagents (Applied Biosystems) from 2 μg unpooledRNA per 100 μl reaction. Each 25-μl amplification reaction contained 10μl template cDNA, 12.5 μl Taqman Universal PCR Master Mix (AppliedBiosystems), 300 nM gene-specific primers and 200 nM gene-specific probe(Table 16). The thermal cycling profile using a ABI StepOnePlusReal-time PCR System (Applied Biosystems) consisted of: Stage 1, onecycle at 50° C. for 2 min; Stage 2, 1 cycle at 95° C. for 10 min; Stage3, 45 cycles at 95° C. for 15 s and 60° C. for 1 min (1 min 30 s forLCT). GAPDH and B-actin mRNA were amplified in duplicate reactions byreal-time PCR from the same reverse transcription reactions as were usedfor the genes of interest. The mean concentration of GAPDH or Beta-actinin each sample was used to control for integrity of input RNA and tonormalize values of target gene expression to those of the housekeepinggene expression. GAPDH and Beta-actin have been shown to be the moststable reference genes in small bowel and colonic biopsies from healthypatients and pediatric patients with inflammatory bowel disease [C1].The final results shown were expressed as the mean copy number fromreplicate biopsies per patient, relative to values obtained for GAPDHmRNA. Beta-actin normalization gave similar results to GAPDHnormalization for all assays (data not shown). Due to insufficient orpoor quality RNA (OD 260/280 ratio <1.7, or RNA integrity number <7.0),only 3 of the 4 biopsies were included for 3 patients (Patient #s 4, 7,10) and only 2 of the 4 biopsies were included for 1 patient (Patient #2). Thus, 83 of the original 88 ileal biopsies were used in real-timePCR experiments.

Lactase genotyping: Genotyping primers for the LCT C/T-13910 andG/A-22018 polymorphisms are as follows: C/T-13910For(5′-GGATGCACTGCTGTGATGAG-3′ [SEQ ID NO: 20]), C/T-13910Rev(5′-CCCACTGACCTATCCTCGTG-3′ [SEQ ID NO: 21]), G/A-22018For(5′-AACAGGCACGTGGAGGAGTT-3′ [SEQ ID NO: 22]), and G/A-22018Rev(5′-CCCACCTCAGCCTCTTGAGT-3′ [SEQ ID NO: 23]). Each 50-μl amplificationreaction contained 500 ng genomic DNA, 400 nM forward and reverseprimers, and 25 μl High Fidelity PCR master mix (Roche). Thermal cyclingconsisted of 1 cycle at 94° C. for 4 min followed by 40 cycles at 94° C.for 1 min, 60° C. for 1 min, and 72° C. for 1 min. PCR reactions forC/T-13910 were directly digested with the restriction enzyme BsmFI at65° C. for 5 hrs. PCR reactions for G/A-22018 were resolved on 1%agarose gels followed by gel extraction of the prominent 448bp amplicon.Gel extracted G/A-22018 amplicons were then digested with therestriction enzyme HhaI at 37° C. for 5 hrs. Restriction digests ofC/T-13910 and G/A-22018 were resolved on 1.5% ethidium-stained agarosegels for genotyping analysis. BsmFI digestion of the C/T-13910 ampliconsgenerates two fragments (351bp and 97bp) for the hypolactasia genotype(C/C), four fragments (351bp, 253bp, 98bp, and 97bp) for theheterozygous genotype (C/T), and three fragments (253bp, 98bp, and 97bp)for the normal homozygous allele (T/T). HhaI digestion of the G/A-22018amplicons generates two fragments (284bp and 184bp) for the hypolactasiagenotype (G/G), three fragments (448bp, 284bp, and 184bp) for theheterozygous genotype (G/A), and a single fragment (448bp) for thenormal homozygous allele (A/A).

Barcoded pyrosequencing of intestinal microbiota: Composite primers usedfor pyrosequencing analysis were as follows: (For)5′-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3′ [SEQ ID NO: 24], (Rev)5′-GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3′ [SEQ ID NO: 25].Underlined sequences in the Forward and Reverse primers represent the454 Life Sciences@ primer B and primer A, respectively. Bold sequencesin the forward and reverse primers represent the broadly-conservedbacterial primer 27F and 338R, respectively. NNNNNNNN represents theeight-base barcode, which was unique for each patient. PCR reactionsconsisted of 8 μl 2.5× 5 PRIME HotMaster Mix (5 PRIME Inc), 6 μl of 4 μMforward and reverse primer mix, and 200 ng DNA in a 20-μl reactionvolume. Thermal cycling consisted of one cycle at 95° C. for 2 min; and30 cycles at 95° C. for 20 seconds, 52° C. for 20 seconds, and 65° C.for 1 min. Each of 4 biopsies per patient was amplified in triplicate,with a single, distinct barcode applied per patient. Ileal and cecalbiopsies were assayed separately. Reagent controls were included(negative controls) to control for any background contamination.Triplicate reactions of individual biopsies and reagent controls werecombined, and PCR products were purified using Ampure magneticpurification beads (Beckman Coulter Genomics) and quantified with theQuanti-iT PicoGreen dsDNA Assay Kit (Invitrogen) and Nanodrop ND-1000Spectrophotometer (Nanodrop Technologies). Equimolar ratios werecombined to create two master DNA pools, one for ileum and one forcecum, with a final concentration of 25 ng/μl. Master pools were sentfor unidirectional pyrosequencing with primer A at 454 Life Sciences ona GS FLX sequencer. Each master pool was sequenced in duplicate ondifferent days to control for variability in the sequencing reactions.Sequences obtained from duplicate runs were combined for the finalanalysis. No sequences were obtained from reagent controls, indicatingthat no background contamination was present.

Quantitative Real-time PCR of Bacteroidete and Firmicute 16S rRNA genes:PCR standards for determining copy numbers of bacterial 16S rDNA wereprepared from representative amplicons of the partial 16S rRNA genes ofBacteroidetes, Firmicutes and total Bacteria cloned into the vectorPGEM-T easy (Promega). A representative amplicon with high sequencesimilarity to Bacteroides Vulgatus (Accession #: NC_(—)009614) was usedwith Bacteroidete-specific primers. A representative amplicon with highsequence similarity to Faecalibacterium prausnitzii (Accession #:NZ_ABED02000023) was used with Firmicute-specific primers. Arepresentative amplicon with high sequence similarity to Bacteroidesintestinalis. (Accession #: NZ ABJL02000007) 16S rRNA gene was used withtotal Bacteria primers (primers 515F and 805R). Cloned sequences wereclassified using the Ribosomal Database Project (RDP, release 10)Seqmatch tool and confirmed by the Microbes BLAST database. Plasmidswere linearized with the SphI restriction enzyme, quantitated, andten-fold serial dilutions of plasmid standards were created ranging from5×10⁷ to 5×10⁰ copies for Bacteroidetes, Firmicutes and total Bacteria.Amplification and detection of DNA by real-time PCR were performed withthe ABI StepOnePlus Real-time PCR System (Applied Biosystems). Cyclingparameters for Bacteroidetes and total Bacteria were as previouslydescribed [C2], as were cycling parameters for Firmicutes [C3]. Each25-μl amplification reaction mixture contained 50 ng DNA, 12.5 μl SYBRGreen Master Mix (Applied Biosystems), and 300 nM bacteria-specific(Bacteroidete, Firmicute or total Bacteria) primers. DNA from each of 88ileal biopsies (4 biopsies per patient) and 88 cecal biopsies (4biopsies per patient) was assayed in duplicate. The final results wereexpressed as the mean number of Bacteroidete or Firmicute 16S rRNA genecopies normalized to 16S rRNA gene copies obtained using total Bacterialprimers. Eight water/reagent controls were included for allamplifications. The average copy number for water/reagent controls(background) was subtracted from each ileal and cecal amplificationprior to normalization. For the Bacteroidete assay all water controlscontained undetectable levels of amplification. For the Firmicute assayaverage amplification signal from water samples were minimal,12.03+/−15.0 copies.

Statistical analysis: To evaluate the effects of CDX2 and/or villin onenzyme and transporter levels and the effects of levels of enzymes andtransporters on bacterial levels, multiple linear regression analyseswere conducted. For assessing the affects of CDX2 and villin ondisaccharidase and transporter expression levels, disaccharidase andtransporter levels were log-transformed to stabilize the variance. Usingeach log-transformed disaccharidase and transporter mRNA expressionlevel as an outcome, three models were fitted: first with CDX2 only asindependent variable; second with CDX2 and status (dummy coded; AUT-GI=1vs. Control-GI=0); and third with CDX2, status, and the interaction termbetween CDX2 and status. The interaction term allowed us to evaluatewhether the effect of CDX2 on disaccharidases and transporters wassimilar for AUT-GI and Control-GI children. The same models were fittedafter adding villin and the interaction term between villin and status.The coefficient estimates in Table 11 represent change inlog-transformed disaccharidase or transporter mRNA levels per unitstandard deviation increase in CDX2 and villin mRNA levels.

To delineate the effects of disaccharidases and transporters onbacterial levels in ileal and cecal biopsies, bacterial 16S rRNA genequantities (obtained from real-time PCR for Bacteroidetes andFirmicutes) or abundance (obtained from 454 pyrosequencing data forProteobacteria and Betaproteobacteria) were log-transformed to stabilizevariance. For each of the log-transformed bacterial levels, enzymelevels were first fitted simultaneously as the main effects (SI, MGAM,LCT, SGLT1, and GLUT2) to evaluate the effects of enzymes on a givenbacterial taxa. Status was added to the model to determine whether therewas a residual difference in bacterial levels between AUT-GI andControl-GI children after adjusting for the levels of disaccharidasesand transporters. It was further examined whether the effect ofdisaccharidases or transporters on bacterial levels was the samedepending on the status by examining two-way interaction terms betweenstatus and each disaccharidase and transporter. The final model wasderived by including all the main effect terms and selectively includingtwo-way interaction terms using the backward elimination method startingfrom all possible two-way interaction terms with status and theindividual disaccharidases and transporters. The coefficient estimatesin Table 14 represent change in log-transformed bacterial levels perunit standard deviation increase in disaccharidase or transporterlevels. The statistical package R (version 2.7.0) was used forregression analysis.

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Example 4 Application of Sutterella-SpecificPCR-based Methods forDetection, Quantitation, and Phylogenetic Characterization of SutterellaSpecies

Abstract. Gastrointestinal disturbances are commonly reported inchildren with autism and can be associated with compositional changes inintestinal bacteria. In a previous report we surveyed intestinalmicrobiota in ileal and cecal biopsies from children with autism andgastrointestinal dysfunction (AUT-GI) and children with onlygastrointestinal dysfunction (Control-GI). The results demonstrated thepresence of members of the family Alcaligenaceae in some AUT-GIchildren, while no Control-GI children had Alcaligenaceae sequences.Here we demonstrate that increased levels of Alcaligenaceae inintestinal biopsies from AUT-GI children result from the presence ofhigh levels of members of the genus Sutterella. We also report the firstSutterella-specific polymerase chain reaction assays for detecting,quantitating, and genotyping Sutterella species in biological andenvironmental samples. Sutterella 16S rRNA gene sequences were found in12 of 23 AUT-GI children but in none of 9 Control-GI children.Phylogenetic analysis revealed a predominance of either the speciesSutterella wadsworthensis or Sutterella stercoricanis in 11 of theindividual Sutterella-positive AUT-GI patients; in one AUT-GI patient,Sutterella-sequences were obtained that could not be given a specieslevel classification based on the 16S rRNA gene sequences of knownSutterella isolates. Western immunoblots revealed plasma IgG or IgMantibody reactivity to Sutterella wadsworthensis antigens in 11 AUT-GIpatients, 8 of whom were also PCR-positive, indicating the presence ofan immune response to Sutterella in some children.

Autism spectrum disorders affect approximately 1% of the population.Many children with autism have gastrointestinal (GI) disturbances thatcan complicate clinical management and contribute to behavioralproblems. Understanding the molecular and microbial underpinnings ofthese GI issues is of paramount importance for elucidating pathogenesis,rendering diagnosis, and administering informed treatment. Anassociation between high levels of intestinal, mucoepithelial-associatedSutterella species and GI disturbances in children with autism isdescribed. These findings elevate this little-recognized bacterium tothe forefront by demonstrating that Sutterella is a major component ofthe microbiota in over half of AUT-GI children and is absent inControl-GI children evaluated in this study. Furthermore, these findingsbring into question the role Sutterella plays in the human microbiota inhealth and disease. With the Sutterella-specific molecular assaysdescribed herein, some of these questions are addressed.

Introduction

Autism spectrum disorders (ASD) are pervasive developmental disordersthat depend on triadic presentation of social abnormalities,communication impairments, and stereotyped and repetitive behaviors fordiagnosis (DSM-IV-TR criteria, American Psychiatric Association, 2000).Gastrointestinal (GI) symptoms are commonly reported in children withautism and can correlate with autism severity (D1, D2). Intestinaldisturbances in autism have been associated with macroscopic andhistological abnormalities, altered inflammatory parameters, and variousfunctional disturbances (D3-9).

In a previous study, we showed that a complex interplay exists betweenhuman intestinal gene expression for disaccharidases and hexosetransporters and compositional differences in the mucoepithelialmicrobiota of children with autism and gastrointestinal disease (AUT-GIchildren) compared to children with GI disease but typical neurologicalstatus (Control-GI children). Significant compositional changes inBacteroidetes, Firmicute/Bacteroidete ratios, and Betaproteobacteria inAUT-GI intestinal biopsies were reported (D10). Although others havedemonstrated changes in fecal bacteria of children with autism (D2,D11-15), the study differed from these by investigating mucoepithelialmicrobiota (D10). The GI microbiota plays an essential role inphysiological homeostasis in the intestine and periphery, includingmaintaining resistance to infection, stimulating immunologicaldevelopment, and perhaps even influencing brain development and behavior(D16-19). Thus, disruption of the balanced communication between themicrobiota and the human host could have profound effects on humanhealth.

In the previous metagenomic study, sequences were found to correspond tomembers of the family Alcaligenaceae in the class Betaproteobacteriathat were present in ileal and cecal biopsies from 46.7% (7/15) ofAUT-GI children. Alcaligenaceae sequences were completely absent frombiopsies of Control-GI children (D10). Members of the familyAlcaligenaceae inhabit diverse habitats, ranging from humans and animalsto soil (D20). Several members of Alcaligenaceae cause clinicallyrelevant infections or are suspected opportunistic pathogens in humansand animals, including members of the genus Bordetella (the humanrespiratory pathogens, B. pertussis and B. parapertussis; the mammalianrespiratory pathogen B. bronchiseptica; and the poultry respiratorypathogen, B. avium); a member of the genus Alcaligenes (the humanopportunistic pathogen A. faecalis); members of the genus Achromobacter(the human opportunistic pathogens A. xylosoxidans and A. piechaudii),members of the genus Oligella (the potential opportunistic genitourinaryspecies O. urethralis and O. ureolytica); a member of the genusTaylorella (the equine urogenital pathogen, T. equigenitalis); and amember of the genus Pelistega (the pigeon respiratory pathogen, P.europaea) (D20).

In some cases the pathogenic potential of Alcaligenaceae members isunclear. The genus Sutterella represents one such Alcaligenaceae member.Members of the genus Sutterella are anaerobic, bile-resistant,asaccharolytic, Gram-negative, short rods (D21). Members of the genusSutterella have been isolated from human infections below the diaphragm(D22, D23). Sutterella 16S rRNA gene sequences have also been identifiedin intestinal biopsies and fecal samples from individuals with Crohn'sdisease and ulcerative colitis (D24, D25). Whether the presence ofSutterella species at sites of human infection and inflammationrepresents cause or consequence, or whether Sutterella is a normal partof the microbiota in some individuals, remains unclear. The dearth ofknowledge concerning the epidemiology and pathogenic potential ofSutterella derives in part from the lack of specific assays to detectand characterize members of this genus.

Alcaligenaceae sequences identified were further characterized in AUT-GIchildren and describe PCR assays for detection, quantitation, andgenotyping of Sutterella as well as serological assays for detection ofimmunological responses to Sutterella.

Results

High levels of Sutterella in a subset of AUT-GI patients identified bypyrosequencing: Previous pyrosequencing results (D10) demonstrated ahigh abundance of sequences from the family Alcaligenaceae in nearlyhalf of AUT-GI children (Patients #1-15) and the absence ofcorresponding sequences in Control-GI children (Patients #16-22), andprompted a more detailed investigation of these taxa of bacteria. Genuslevel analysis of pyrosequencing reads revealed that all sequences ofAlcaligenaceae found in AUT-GI patients' biopsies were classified asmembers of the genus Sutterella. The average confidence estimate of allgenus level, RDP (Ribosomal Database Project)-classifiedSutterella-sequences was high (99.1%), with the majority of sequencesclassified at 100% confidence.

Comparison of Sutterella abundance from pyrosequencing reads revealedsignificant increases in Sutterella in the ilea (FIG. 8A: Mann-Whitney,tied p-value=0.022) and ceca (FIG. 8B: Mann-Whitney, tied p-value=0.037)of AUT-GI children compared to Control-GI children. Individual analysisof AUT-GI patients revealed that 46.7% ( 7/15) of AUT-GI patients(Patients # 1, 3, 5, 7, 10, 11, 12) had high levels of Sutterella 16SrRNA gene sequences in both the ileum (FIG. 8C and Table 19) and cecum(FIGS. 8D and 36 and Table 19). Sutterella-sequences were absent fromall Control-GI samples (Patients # 16-22). In those seven AUT-GIpatients with Sutterella-sequences, ileal Sutterella-sequence abundanceranged from 1.7 to 6.7% of total bacterial reads (FIG. 8C and Table 19).For the same patients, cecal Sutterella-sequence abundance ranged from2.0 to 7.0% of total bacterial reads (FIGS. 8D and 36 and Table 19).

TABLE 19 Summary of total bacteria, Betaproteobacteria, and Sutterellasequences obtained by 16S rRNA gene (V2-region) pyrosequencing fromileal and cecal biopsies of AUT-GI and Control-GI children. # of Total #of Total # of Beta- Bacteria Bacteria proteobacteria Patient #AUT/Control Reads-Ileum Reads-Cecum Reads-Ileum 1 AUT-GI 11,881 13,032706 2 AUT-GI 13,734 7,647 3627 3 AUT-GI 11,434 10,147 536 4 AUT-GI12,756 11,779 400 5 AUT-GI 10,708 10,502 647 6 AUT-GI 14,739 11,075 1377 AUT-GI 11,941 11,246 209 8 AUT-GI 11,348 11,754 27 9 AUT-GI 12,32010,661 262 10 AUT-GI 12,483 12,295 501 11 AUT-GI 11,211 12,436 800 12AUT-GI 11,055 11,103 434 13 AUT-GI 10,420 10,670 171 14 AUT-GI 12,21711,012 123 15 AUT-GI 12,002 12,561 138 16 Control-GI 13,758 13,630 12917 Control-GI 12,246 14,956 147 18 Control-GI 11,888 14,330 315 19Control-GI 11,290 10,136 377 20 Control-GI 14,844 11,794 134 21Control-GI 13,308 11,567 145 22 Control-GI 13,460 10,143 131 # of # ofSutterlla-Ileum Sutterella-Ileum Betaproteo- Sutterella (% of (% ofPatient bacteria Reads- Total Betaproteo- # Reads-Cecum Ileum Bacteria)bacteria) 1 535 534 4.5 75.6 2 632 0 0 0 3 428 503 4.4 93.8 4 132 0 0 05 535 581 5.4 89.8 6 36 0 0 0 7 224 201 1.7 96.2 8 80 0 0 0 9 404 0 0 010 478 490 3.9 97.8 11 903 747 6.7 93.4 12 444 408 3.7 94.0 13 619 0 0 014 105 0 0 0 15 39 0 0 0 16 136 0 0 0 17 116 0 0 0 18 404 0 0 0 19 151 00 0 20 58 0 0 0 21 34 0 0 0 22 85 0 0 0 # of Sutterella- Sutterella-Sutterella Cecum (% of Cecum (% of Patient # Reads-Cecum Total Bacteria)Betaproteobacteria) 1 520 4.0 97.2 2 0 0 0 3 403 4.0 94.2 4 0 0 0 5 4984.7 93.1 6 0 0 0 7 220 2.0 98.2 8 0 0 0 9 0 0 0 10 459 3.7 96.0 11 8707.0 96.3 12 409 3.7 92.1 13 0 0 0 14 0 0 0 15 0 0 0 16 0 0 0 17 0 0 0 180 0 0 19 0 0 0 20 0 0 0 21 0 0 0 22 0 0 0

To put the levels of Sutterella in these patients into perspective, theabundance of all ileal and cecal genus level classifications were rankedfrom pyrosequencing results. In the ileum, Sutterella-sequencesrepresented the 4^(th) most abundant genera for patient #1, the 6^(th)most abundant genera for patient #3, the 5^(th) most abundant genera forpatient #5, the 5^(th) most abundant genera for patient #7, the 3^(rd)most abundant genera for patient #10, the 8^(th) most abundant generafor patient #11, and the 5^(th) most abundant genera for patient #12(FIG. 44 and FIG. 45). Similar rankings were obtained in the cecum ofthese patients.

Sutterella-sequences represented the majority of sequences present inthe class Betaproteobacteria in these seven AUT-GI patients. In ilealbiopsies from the seven AUT-GI patients with Sutterella-sequences,Sutterella-sequences accounted for 75.6% to 97.8% of allBetaproteobacteria sequences (FIG. 8E and Table 19). In cecal biopsies,Sutterella-sequences accounted for 92.1% to 98.2% of allBetaproteobacteria sequences (FIG. 8F and Table 19).

OTU and sequence analysis of Sutterella-sequences in AUT-GI children:OTU (Operational Taxonomic Unit) analysis of V2 pyrosequencing reads inileum (FIG. 46A) and cecum (FIG. 46B) revealed that sequences frompatients #1, 3, 10, 11, and 12 clustered together with OTU 2 containingthe majority of Sutterella-sequences, and patients #5 and 7 clusteredtogether with OTU 1 containing the majority of Sutterella-sequences. OTU2 accounted for 87% and 84% for patient #1, 85% and 87% for patient #3,66% and 66% for patient #10, 87% and 85% for patient #11, and 81% and81% for patient #12 of all Sutterella-sequences obtained bypyrosequencing of the 16S rRNA gene in ileum and cecum, respectively(FIG. 37). OTU 1 accounted for 88% and 86% for patient #5 and 88% and83% for patient #7 of all Sutterella sequences obtained bypyrosequencing of the V2 region of the 16S rRNA gene in ileum and cecum,respectively (FIG. 37). Subdominant OTUs can represent true phylotypes,but could also arise from PCR or sequencing artifacts. The analysis wasfocused on those OTUs containing the majority of Sutterella-sequences,namely OTU 1 and OTU 2.

The representative sequences from OTU 1 and OTU 2 were aligned and usedfor phylogenetic analysis (FIG. 47. The representative sequence from OTU1 was phylogenetically most closely related to the species S.wadsworthensis; the representative sequence from OTU 2 was most closelyrelated to S. stercoricanis. Although some branches in the tree areclearly differentiated by high bootstrap values, others aredifferentiated poorly by low bootstrap values. Furthermore, members ofthe genus Comamonas and Burkholderia were grouped with members of thegenus Sutterella. This indicates that sequences from the V2 region alonecan be insufficient for accurate species level phylogenetic analysis ofSutterella-sequences.

Confirmation and quantitation of Sutterella-sequences using new PCRassays: To independently verify V2 pyrosequencing results forSutterella, Sutterella-specific PCR assays were designed that could beused in both conventional and real-time PCR, using primers that amplifya 260bp region spanning the V6 to V8 regions of the 16S rRNA gene(SuttFor and SuttRev primers) (FIG. 38, FIG. 9, FIG. 10A-B).Conventional PCR analysis using DNA from each of 4 ileal and 4 cecalbiopsies per patient showed that the same individuals identified ashaving high levels of Sutterella by V2 pyrosequencing (Patients #1, 3,5, 7, 10, 11, 12) were also positive by the novel V6-V8Sutterella-specific PCR (FIG. 39A). All 4 biopsies from ileum and cecum,in all seven Sutterella-positive patients, showed Sutterella products. Asingle 260 by product was observed in positive amplifications, andnon-specific products were never observed. No products were observed inany Control-GI patients that were evaluated by pyrosequencing (Patients#16-22), the AUT-GI patients that were negative for Sutterella sequencesby V2 pyrosequencing (Patients #2, 4, 6, 8, 9, 13, 14, 15), orwater/reagent controls (FIG. 39A). Furthermore, the positive control(DNA from a cultured S. wadsworthensis isolate) was positive by PCR. Inaddition to those patients evaluated by pyrosequencing, ileal and cecalbiopsies were assessed from eight additional male AUT-GI (Patients#23a-30a) and two additional male Control-GI (Patients #31a and 32a)children using the V6-V8 Sutterella PCR. Of these additional samples, 5of the 8 AUT-GI patients were positive for Sutterella in ileal and cecalbiopsies (Patients #24a, 25a, 27a, 28a, and 29a). All biopsies from thetwo additional Control-GI patients were PCR-negative (Patients #31a and32a). In summary, whereas 12 of 23 (52%) AUT-GI children werePCR-positive for Sutterella, 0 of the 9 Control-GI children werePCR-positive for Sutterella.

In addition, the broadly conserved, pan-bacterial primer 515For was usedin combination with the SuttRev primer in conventional PCR assays (FIG.39B). These primers amplify a 715 by region of the 16S rRNA gene fromconserved region 4 (C4) to variable region 8 (V8) (see FIG. 38A).Results of the C4-V8 amplification were identical to the V6-V8 assay.All products were confirmed to represent Sutterella by sequencing ofV6-V8 and C4-V8 products. These results indicate that the SuttRev primeris sufficient to confer specificity for Sutterella amplification.

In addition, Sutterella 16S rRNA gene sequences were quantified inbiopsies from AUT-GI and Control-GI patients using real-time PCR (FIG.10A-B). Real-time PCR analysis using the SuttFor and SuttRev (V6-V8)primers and a high coverage Taqman probe revealed similar results toconventional PCR assays. By real-time PCR, Sutterella was detected inpatients #1, 3, 5, 7, 10, 11, 12, 24a, 25a, 27a, 28a, and 29a (FIG. 40),consistent with both pyrosequencing and conventional PCR results.Sutterella was undetectable in all Control-GI and Sutterella-negativeAUT-GI patients' samples. Mean Sutterella copy numbers were high in boththe ileum and cecum [in the range of 10³ to 10⁵ copies] ofSutterella-positive patients.

Phylogenetic analysis of Sutterella-sequences obtained by novel PCRassays: The predominant Sutterella-sequence from the ileum and cecum ofeach patient was determined following alignment of all V6-V8 sequencesobtained by library cloning of products. This analysis revealed that thepredominant sequences obtained in ileal biopsies were identical to thepredominant sequences in cecal biopsies from each individual patient.Thus, a single predominant sequence was further assessed for eachpatient.

Phylogenetic analysis of the predominant V6-V8 sequences obtained by PCRrevealed that the dominant Sutterella species found in patients #1, 3,10, 11, 12, 24a, 27a, and 29a were most closely associated with theisolates S. stercoricanis and Parasutterella secunda; the dominant V6-V8Sutterella-sequences found in patients #5, 7, and 25a were most closelyassociated with isolates of S. wadsworthensis (FIG. 48). Sequences frompatient #28a were most closely associated with Sutterella sp. YIT 12072.Thus, sequences from patients #5 and 7. grouped with S. wadsworthensisisolates using both the V2 pyrosequencing reads and the V6-V8 sequencesobtained by PCR, while sequences from patients #1, 3, 10, 11, and 12grouped with S. stercoricanis using both the V2 pyrosequencing reads andthe V6-V8 sequences obtained by PCR. However, as was the case fromphylogenetic analysis of V2 pyrosequencing, bootstrap values were low atmany branches, indicateing that neither the V2 nor V6-V8 regions providesufficient information for accurate species level differentiation.Furthermore, members of the genus Sutterella did not all group togetherbased on the V6-V8 region sequences, with Parasutterella secunda, S.stercoricanis, S. sanguinus, and S. morbirenis being more closelyassociated with other Alcaligenaceae and Burkholderiales members.

480 sequences (40 sequences per patient; 20 ileal sequences and 20 cecalsequences) obtained from clone libraries of C4-V8 products were analyzedfrom the 12 Sutterella-positive patients (FIG. 41). No sequences wereobtained from any genus other than Sutterella from any cloned PCRproducts. The majority or all of the C4-V8 sequences from patients #1,3, 10, 11, 12, 24a, 27a, and 29a were most closely matched with S.stercoricanis, the majority or all of the C4-V8 sequences obtained frompatients #5, 7, and 25a matched most closely with S. wadsworthensis, andall sequences obtained from patient #28a matched most closely withSutterella sp. YIT 12072. It was evident from this analysis thatalthough one species predominated in each patient, mixed populationswere detected in many patients. Most individuals with mixed populationsharbored sequences of S. wadsworthensis and S. stercorcanis. Patient#24a had species matches for S. stercoricanis, S. wadsworthensis, and S.parvirubra.

To determine the accuracy of the C4-V8 region for confirmation ofspecies level classification, the predominant C4-V8 16S rRNA genesequences obtained from the ileum and cecum of each patient wereanalyzed. Similar to the results obtained with the V6-V8 region, thisanalysis revealed that the predominant Sutterella 16S rRNA genesequences identified in ileal biopsies were identical to the predominantSutterella-sequences in cecal biopsies for each of the individualpatients. Thus, a single predominant sequence was further assessed foreach patient.

Alignment of the predominant C4-V8 sequence from each patient revealedthat patients #1 and 24a had identical predominant sequences, but thatthese were distinct from all other patients; patients #3, 10, 11, 12,27a, and 29a had identical sequences, distinct from all other patients;patients #5, 7, and 25a had identical sequences that were distinct fromall other patients; and patient #28a had a unique sequence (FIG. 49).

Comparison of percent sequence similarity between these groups (Table17) revealed 99.9% similarity between sequences of patients #1 and 24aand those of patients #3, 10, 11, 12, 27a, and 29a. This value is abovethe cut-off value of 97% similarity, commonly applied for bacterialspecies definition (D26), indicateing that the predominant sequencesfrom these two groups are likely the same species.

TABLE 17 Sequence similarity between 16S rRNA gene (C4-V8 region) ofSutterella from AUT-GI children and Sutterella isolates. PatientsSutterella Sunerella Sutterella Sutterella Sutterella sp. Patients 3,10, 11, 12, Patients Patient stercoricanis wadsworthensis parvirubrasanguinus YIT 12072 % Similarity 1, 24a 27a, 29a 5, 7, 25a 28a(AJ566849) (GU585669) (AB300989) (AJ748647) (AB491210) Patients — 99.9%94.8% 93.8% 98.5% 94.8% 95.4% 96.3% 93.2% 1, 24a Patients — 94.7% 93.6%98.4% 94.7% 95.4% 96.4% 93.3% 3, 10, 11, 12, 27a, 29a Patients — 92.8%94.7%  100% 96.6% 93.6% 92.9% 5, 7, 25a Patient — 93.0% 92.8% 93.6%92.0% 95.3% 28a Sutterella — 94.7% 94.7% 96.6% 93.2% stercoricanis(AJ566849) Sutterella — 96.6% 93.6% 92.9% wadsworthensis (GU585669)Sutterella — 95.0% 92.6% parvirubra (AB300989) Sutterella — 92.2%sanguinus (AJ748647) Sutterella sp. — YIT 12072 (AB491210) Highestsequence similarities are shown in bold.

The predominant sequences from patients #1 and 24a and patients #3, 10,11, 12, 27a, and 29a had the highest percent similarity to the isolateS. stercorcanis (98.5% similarity and 98.4% similarity, respectively)(Table 17). The percent similarity of sequences from patients #1, 24a,3, 10, 11, 12, 27a, and 29a were below 97% compared to the otherSutterella isolates, indicateing that the predominant species in thesepatients is likely S. stercorcanis. In addition, the 16S rRNA genesequence from patients #1 and 24a shared 100% similarity with 16S rRNAgene sequences from uncultured bacteria in genbank, such as thosederived from intestinal biopsies from an ulcerative colitis patient(i.e., Accession FJ512128) (D27) and mucosal biopsies from theintestinal pouch of a familial adenomatous polyposis patient (i.e.,Accession GQ159316). Similarly, the sequences from patients #3, 10, 11,12, 27a, and 29a shared 100% similarity with 16S rRNA gene sequencesfrom uncultured bacteria in genbank, including sequences derived fromintestinal biopsies from a patient with ulcerative colitis (i.e.,Accession 512152) (D27) and fecal samples from bovines (i.e., AccessionFJ682648) (D28).

Sequences from patients #5, 7, and 25a had 100% sequence similarity toS. wadsworthensis and below 97% sequence similarity to all otherSutterella isolates (Table 17). Thus, the predominant sequences frompatients #5, 7, and 25a are likely S. wadsworthensis. The sequence frompatients #5, 7, and 25a also shared 100% sequence similarity to 16S rRNAsequences in genbank, such as those derived from intestinal biopsiesfrom an ulcerative colitis patient (i.e., Accession FJ509042) (D27).

The unique sequence found in patient #28a matched most closely with theisolate, Sutterella sp. YIT 12072; however, the percent similarity wasonly 95.3% (Table 17). Thus, based on sequence analysis alone,Sutterella-sequences from patient #28a cannot be classified asSutterella sp. YIT 12072 or any of the other known isolates. Despite theclosest association of sequences from patient #28a with the sequence ofthe isolate Sutterella sp. YIT 12072, the 16S rRNA gene sequence frompatient #28a shared 100% similarity with 16S rRNA gene sequences fromuncultured bacteria in genbank that were derived from intestinalbiopsies from a Crohn's disease'patient (i.e., Accession FJ503635)(D27), human skin popliteal fossa swab (i.e., Accession HM305996), andfeces from a 95-year old woman (i.e., Accession EF401376) (D29). Thus,the 16S rRNA gene sequences from patient #28a and identical genbanksequences likely represent an uncharacterized species of Sutterella.

Phylogenetic analysis of the predominant sequences obtained from patientbiopsies using the C4-V8 PCR assay revealed high bootstrap values atmost branches and good grouping of members of the genus Sutterella fromother Alcaligenaceae family members and other Burkholderiales ordermembers (FIG. 42). Thus, sequences obtained by C4-V8 PCR can be used foraccurate species level classification of Sutterella-sequences. This treedemonstrates that sequences from patients #1, 24a, 3, 10, 11, 12, 27a,and 29a grouped most closely with S. stercoricanis (supported by abootstrap resampling value of 92%); sequences from patients #5, 7, and25a grouped most closely with S. wadsworthensis (supported by abootstrap resampling value of 99%); and sequences from patient #28agrouped most closely with the isolate Sutterella sp. YIT 12072(supported by a bootstrap resampling value of 97%) but formed a distinctphylogenetic lineage.

AUT-GI plasma antibodies bind to S. wadsworthensis proteins: It was alsodetermined whether systemic antibody responses to Sutterella werepresent in this cohort. The antigens used for western blot analysis werewhole protein lysates from cultured S. wadsworthensis containing a widerange of proteins, as observed on Coommassie-stained SDS-polyacrylamidegels. Individual patient's plasma was assessed for IgG (FIG. 43A) andIgM (FIG. 43B) antibody immunoreactivity against the bacterial antigens.Immunoreactive bands were visible for 11 out of 23 (48%) AUT-GIpatients. In ten AUT-GI children the immunoreactive antibodies were IgG(FIG. 43A); one (patient #26a) had IgM antibodies (FIG. 43B). Incontrast, only 1 of the 9 (11%) Control-GI patients (patient #21) hadweak immunoreactivity to 84-kDa and 41-kDa Sutterella proteins. A totalof 11 distinct immunoreactive protein bands were identified, based onsize (104-, 89-, 84-, 62-, 56-, 50-, 48-, 44-, 41-, 30-, and 27-kDa).AUT-GI patients #1 and #5 (both positive by PCR) had the mostimmunoreactive protein bands with four protein bands in common (89-,62-, 56-, and 41-kDa). The 89-kDa band was detected by IgG or IgMantibodies in seven AUT-GI patients. The 56-, 41-, and 30-kDa bands weredetected by IgG antibodies in each of three patients. The other bands(104-, 84-, 62-, 50-, 48-, and 44-kDa) were less frequent.

Of the 12 AUT-GI patients that were PCR-positive for Sutterella, 8(66.7%) had plasma IgG antibodies against S. wadsworthensis proteins(patients #1, 3, 5, 7, 10, 11, 24a, and 25a). Three AUT-GI patients(patients #4, 23a, and 26a) had IgG or IgM antibodies against S.wadsworthensis proteins, but were PCR-negative. In total, 15 out of 23(65.2%) AUT-GI children had evidence of Sutterella either by PCR orserology (Table 18).

TABLE 18 Summary of PCR assays and western immunoblot analysis. PatientAUT/Control PCR IgG MW of Bands IgM MW of Bands Any Ig Positive AnyPositive 1 AUT-GI + ++ 89, 62, 56, 41 − − Yes Yes 2 AUT-GI − − − − − NoNo 3 AUT-GI + + 30 − − Yes Yes 4 AUT-GI − ++ 89 − − Yes Yes 5 AUT-GI +++ 89, 62, 56, 48, 44, 41 − − Yes Yes 6 AUT-GI − − − − − No No 7AUT-GI + ++ 50, 44 − − Yes Yes 8 AUT-GI − − − − − No No 9 AUT-GI − − − −− No No 10  AUT-GI + ++ 30 − − Yes Yes 11  AUT-GI + + 89, 48 − − Yes Yes12  AUT-GI + − − − − No Yes 13  AUT-GI − − − − − No No 14  AUT-GI − − −− − No No 15  AUT-GI − − − − − No No 23a AUT-GI − ++ 104, 30, 27 − − YesYes 24a AUT-GI + + 89 − − Yes Yes 25a AUT-GI + ++ 89, 56 − − Yes Yes 26aAUT-GI − − − ++ 89 Yes Yes 27a AUT-GI + − − − − No Yes 28a AUT-GI + − −− − No Yes 29a AUT-GI + − − − − No Yes 30a AUT-GI − − − − − No No 16 Control-GI − − − − − No No 17  Control-GI − − − − − No No 18  Control-GI− − − − − No No 19  Control-GI − − − − − No No 20  Control-GI − − − − −No No 21  Control-GI − + 84, 41 − − Yes Yes 22  Control-GI − − − − − NoNo 31a Control-GI − − − − − No No 32a Control-GI − − − − − No No %AUT-GI+ 52% 43% − 4% − 48% 65% % Control-GI+  0% 11% − 0% − 11% 11%

Discussion

Detection by pyrosequencing of Alcaligenaceae sequences in AUT-GIchildren (10) was previously reported. More focused analysis revealedthat this finding reflects the presence of Sutterella species. Whereas12 of 23 AUT-GI patients (52%) were PCR-positive both in ileum andcecum, 0 of 9 Control-GI children'were PCR-positive for Sutterella.Sutterella abundance in the seven Sutterella-positive AUT-GI patients,assessed by pyrosequencing, ranged from 1 to 7% of total bacterialsequences. Novel real-time PCR assays confirmed high copy numbers ofSutterella species in DNA from ileal and cecal biopsies ofSutterella-positive patients, with averages ranging from 10³ to 10⁵Sutterella 16S rRNA gene copies amplified from only 25 ng of totalgenomic biopsy DNA.

OTU analysis of V2-region pyrosequencing reads indicated that only twoOTUs accounted for the majority of Sutterella-sequences in the sevenAUT-GI patients that were Sutterella-positive by pyrosequencing.Sequencing of PCR products from V6-V8 and C4-V8 Sutterella-specific PCRassays corroborated this finding. The analysis also indicates that C4-V8Sutterella products can be accurately classified at the species level.Classification with RDP and phylogenetic analysis ofSutterella-sequences obtained from C4-V8 Sutterella-specific PCRindicated that the predominant sequences obtained from patients #1, 3,10, II, 12, 24a, 27a, and 29a were most closely related to the isolateS. stercoricanis, supported by a sequence similarity of over 98%. Thepredominant C4-V8 sequences obtained from patients #5, 7, and 25a weremost closely related to the isolate S. wadsworthensis, supported by asequence similarity of 100%. The results indicate that these two speciesof Sutterella are the dominant phylotypes present at high levels in theintestines of AUT-GI children in this cohort. Of the known isolates, thepredominant C4-V8 sequence obtained from patient #28a was most closelyrelated to Sutterella sp. YIT 12072. However, the low sequencesimilarity (95.3%) between sequences from patient #28a and Sutterellasp. YIT 12072 indicates that these are not likely to be the samespecies. Sequences from patient #28a did have 100% sequence similaritywith uncultured Sutterella-sequences in genbank, indicating that thisundefined species has been detected previously in human samples usingnon-specific techniques.

Sutterella species have been isolated from human and animal feces(D30-D32) and have also been isolated from human infections below thediaphragm; most often from patients with appendicitis, peritonitis orrectal or perirectal abscesses (D22, D23). Sutterella-sequences havebeen identified in fecal samples and intestinal biopsies fromindividuals with Crohn's disease and ulcerative colitis but also fromapparently healthy adults (D24, D25, D27, D33). Without being bound bytheory, Sutterella species can contribute to inflammation and infectionor are simply normal inhabitants of the human microbiota in someindividuals. Even if the latter is the case, the results demonstratethat Sutterella is a major component of the mucoepithelial microbiota insome children, accounting for up to 7% of all bacteria. Relative to allother bacterial genera identified in biopsies, Sutterella ranged fromthe 3^(rd) to 8^(th) most abundant genera in the patients assessed bypyrosequencing. Only the most abundant Bacteroidete and Firmicute generaoutnumbered Sutterella-sequences. This result is remarkable given thatSutterella is not reported as a major component of the microbiota (D34).

Loss of commensals in the intestine can affect immune responses anddisrupt colonization resistance to potentially pathogenic bacteria (D17,D19). A significant loss of commensals, namely members of theBacteroidete phyla, were found in AUT-GI biopsies (D10). Thus, the lossof Bacteroidetes in AUT-GI children could facilitate the growth ofopportunistic pathogens. Whether Sutterella is pathogenic in AUT-GIchildren cannot be determined from current data. However, theobservation that some AUT-GI children have antibodies that react with S.wadsworthensis proteins is generally consistent with infection. Wedetected either IgG or IgM antibodies against S. wadsworthensis proteinsin approximately 48% (11/23) of AUT-GI children. Only one Control-GIchild had very weak IgG immunoreactivity against S. wadsworthensisproteins. Of the 12 patients that were positive for Sutterella by PCR, 8(66.7%) demonstrated plasma IgG antibodies against S. wadsworthensisproteins. In total, 65.2% (15 out of 23) of AUT-GI children were eitherpositive by PCR assays or had immunoglobulin reactivity to S.wadsworthensis proteins. Three AUT-GI patients were negative by PCR buthad IgG or IgM antibodies against S. wadsworthensis proteins. Withoutbeing bound by theory, Sutterella species can also be present in otherregions of the small or large intestine or elsewhere in the body ofthese three patients, explaining the presence of Sutterella-specificantibodies without detection of the agent by PCR. Alternatively, IgGantibodies can persist long after antigenic exposure; thus, the presenceof IgG antibodies can indicate past exposure in some children. The IgMimmunoreactivity of patient #26a indicates recent or current exposure toSutterella antigen in this patient. It is well recognized that the useof different strains and species as antigen leads to variations in theimmunoreactive profile of immunogenic proteins (D35). SeveralSutterella-positive patients in this study had S. stercoricanis as thedominant Sutterella species.

The nature of intestinal damage in autism has not been fully defined.Abnormalities in intestinal permeability in children with autism havebeen reported in two studies (D8, D9). In Crohn's disease, a conditionassociated with increased intestinal permeability, a generalizedenhancement of antimicrobial IgG to many members of the intestinalmicrobiota is reported (D36). A defective epithelial barrier could leadto enhanced contact between many members of the microbiota andantigen-presenting cells in the lamina propria. If this turns out to bethe case in autism, then antibodies against Sutterella proteins canreflect inter-individual, compositional variation in the microbiota,rather than an indication of Sutterella infection.

In conclusion, Sutterella 16S rRNA gene sequences were identified inmucoepithelial biopsies from AUT-GI children using non-specific,pan-microbial pyrosequencing. New Sutterella-specific PCR assays weredesigned and applied that confirmed high levels of Sutterella species inover half of AUT-GI children and the complete absence of Sutterella inControl-GI children tested in this study. The Sutterella-specificmolecular assays reported in this study will enable more directedstudies to detect, quantify, and classify this poorly understoodbacterium in biological and environmental samples. With such specifictechniques, the following can be understood: the epidemiology of thisbacterium and its associations with human infections and inflammatorydiseases; the role Sutterella plays in the microbiota, and the extent towhich Sutterella can contribute to the pathogenesis of GI disturbancesin children with autism.

Materials and Methods

Clinical samples: Clinical procedures for this study population arepreviously described (D10, D37). The Institutional Review Board (IRB) atColumbia University Medical Center reviewed and approved the use ofde-identified residual ileal and cecal samples, obtained as described inan earlier publication (D37), and waived the need for patient consentfor these analyses, as all samples were analyzed anonymously. Patientsassessed by pyrosequencing were restricted to male children between 3 to5 years of age to control for confounding effects of gender and age onthe microbiota (D10). This subset comprised 15 AUT-GI (patients #1-15)and 7 Control-GI (patients #16-22) children. For assessment ofSutterella-sequences in ileal and cecal biopsies, we also included 8additional male AUT-GI children (patients #23a-30a: 6 children between 6and 7 years of age, and 2 children between 8 and 10 years of age) and 2additional male Control-GI children (patients #31a and 32a: 1 childbetween 6 and 7 years of age and 1 child between 8 and 10 years of age)from the initial cohort (D37).

Bacterial Culture: S. wadsworthensis was obtained from American TypeCulture Collection (ATCC, # 51579). The isolate was grown in choppedmeat broth in Hungate capped tubes (Anaerobe Systems, Morgan Hill,Calif.), supplemented with sodium formate and fumaric acid at a finalconcentration of 0.3% each. Inoculated cultures were incubated at 37° C.and growth was monitored at 0, 6, 12, 24, and 48 hours using aSutterella-specific real-time PCR assay (see below).

DNA extraction: DNA was extracted from individual ileal and cecalbiopsies (total of 256 biopsies: 128 ileal biopsies and 128 cecalbiopsies; 8 biopsies per patient [4 from ileum and 4 from cecum]; 23AUT-GI patients and 9 Control-GI patients) and bacterial cultures of S.wadsworthensis in TRIzol (Invitrogen, Carlsbad, Calif.) using standardprotocols. DNA concentrations and integrity were determined using aNanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington,Del.) and Bioanalyzer (Agilent Technologies, Foster City, Calif.) andstored at −80° C.

Pyrosequencing: Barcoded pyrosequencing of the bacterial V2 region ofthe 16S rRNA gene and analyses are previously described for ileal andcecal biopsies from AUT-GI patients #1-15 and Control-GI patients #16-22(D10). The pan-bacterial barcoded V2 primers, designated V2For andV2Rev, amplify a region of the 16S rRNA gene from nucleotide position 27to 338 (D38) (FIG. 38).

Sutterella-specific PCR assay design: Sutterella-specific 16S rRNA PCRprimers were designed against the 16S rRNA gene sequence for S.wadsworthensis (Accession L37785) using Primer Express 1.0 software(Applied Biosystems, Foster City, Calif.). Genus specificity ofcandidate primers was evaluated using the RDP (Ribosomal DatabaseProject) probe match tool. Several potential primer pairs wereidentified but only one pair showed high specificity for Sutterella.These primers are designated here as SuttFor (nucleotide position936-956 of S. wadsworthensis: Accession L37785) and SuttRev (nucleotideposition 1177-1195 of S. wadsworthensis: Accession L37785) [Table 20].SuttFor and SuttRev primers amplify a 260 base pair (bp) region betweenvariable regions 6, 7 and 8 (V6-V8) of the 16S rRNA gene of Sutterella(FIG. 38).

TABLE 20 Primers and probes used for conventional PCR or real-timePCR amplification and quantitation of Sutterella species. NucleotideAmplicon SEQ Name Primers and Probe (5′-3′) Position* size (bp) ID NO:Sutterella SuttFor: CGCGAAAAACCTTACCTAGCC 936-956 ~260 11 (V6-V8)SuttRev: GACGTGTGAGGCCCTAGCC 1177-1195 12 SuttProbe:FAM-CACAGGTGCTGCATGGCTGTCGT-NFQ 1011-1033 13 Sutterella 515For:GTGCCAGCMGCCGCGGTAA 482-500 ~715 65 (C4-V8) SuttRev: GACGTGTGAGGCCCTAGCC1177-1195 66 Total 515For: GTGCCAGCMGCCGCGGTAA 482-500 ~292 15 Bacteria805Rev: GACTACCAGGGTATCTAAT 754-772 16 *Nucleotide position relative tothe 16S rRNA gene of Sutterella wadsworthensis (Accession # L37785)

Conventional PCR Assays: Conventional PCR for detection of Sutterellawas carried out in 25 μl reactions consisting of 25 ng of biopsy DNA or25 pg of genomic DNA from cultured S. wadsworthensis (ATCC, # 51579:positive control), 300 nm each SuttFor and SuttRev primers (for V6-V8amplification) or 515For and SuttRev (for C4-V8 amplification), 2 μldNTP Mix (10 mM; Applied Biosystems, Foster City, Calif.), 2.5 μl of10×PCR Buffer (Qiagen, Valencia, Calif.), 5 U of HotStarTaq DNApolymerase (Qiagen), and 5 μl Q-solution (Qiagen). Cycling parametersconsisted of an initial denaturation step at 95° C. for 15 min, followedby 30 cycles of 94° C. for 1 min, 60° C. for 1 min, 72° C. for 1 min,and a final extension at 72° C. for 5 min. The amplified product wasdetected by electrophoresis on a 1.5% agarose gel stained with ethidiumbromide. To confirm specificity of PCR amplification, V6-V8 productswere gel-extracted and sent for direct sequencing with SuttFor andSuttRev primers. Additionally, V6-V8 and C4-V8 products were subclonedinto the vector PGEM-T easy (Promega, Madison, Wis.) and bacteriallibraries were created. One hundred and twenty V6-V8 plasmid clones weresequenced. A total of 480 C4-V8 colonies were sequenced and analyzed (40sequences from each of the 12 PCR-positive patients; 20 sequences fromileal and 20 sequences from cecal biopsies). All V6-V8 and C4-V8 plasmidclones were found to contain Sutterella-sequences using the RDPclassifier tool with a minimum 80% bootstrap confidence estimate. Theclosest sequence match to Sutterella isolates was determined using theRDP seqmatch tool. Sequences from each individual patient were alignedusing MacVector, and a consensus sequence was determined from thepredominant Sutterella species in each patient.

Quantitative Real-time PCR Assay: PCR standards for determining copynumbers of bacterial 16S rRNA genes were prepared from products of thepartial 16S rRNA gene (V6-V8 region) of S. wadsworthensis (AccessionGU585669). A representative product with high sequence similarity toBacteroides intestinalis (Accession NZ_ABJL02000007) 16S rRNA gene wasused with broadly conserved total bacteria primers (D10, D39). Productswere cloned into the vector PGEM-T easy (Promega) and ten-fold serialdilutions of linearized plasmid standards were created ranging from5×10⁵ to 5×10⁰ copies. Amplification and detection of DNA by real-timePCR were performed with the ABI StepOne Plus Real-time PCR system(Applied Biosystems). Linearity and sensitivity of plasmid standardswere tested with SuttFor and SuttRev primers and the SuttProbe.Amplification plots of plasmid standards indicated sensitivity ofdetection down to 5 copies of plasmid (FIG. 10A), and standard curvesgenerated from plasmid dilutions had correlation coefficients of 0.996(FIG. 10B).

Bioinformatics analysis: Operational taxonomic unit (OTU)-based analysisof pyrosequencing data was carried out in MOTHUR (version 1.8.0) and aspreviously described (D10, D40).

Phylogenetic analysis of Sutterella sequences: Phylogenetic analyseswere conducted in MEGA4 (D41). Sequence alignments were based onrepresentative sequences from OTU 1 and OTU 2, obtained frompyrosequencing analysis of the V2 region of the 16S rRNA gene, as wellas sequences of Sutterella from the V6-V8 (SuttFor and Sutt Revamplifications) conventional PCR assay, and the predominant sequencesobtained from clone libraries of the C4-V8 (515For and SuttRevamplifications) conventional PCR assay. Primer sequences were trimmedfrom the sequences. Classification was confirmed using the RDPclassifier and seqmatch tools. Sutterella sequences obtained from ilealand cecal biopsies were aligned with sequences from 8 isolates ofSutterella found in the RDP database and sequences from 14 additionalrelated species (members of the family Alcaligenaceae and orderBurkholderiales). Sequences from Sutterella isolates and related specieswere trimmed to the length of the sequences obtained from ileal andcecal biopsies of AUT-GI patients. Phylogenetic trees were constructedaccording to the neighbour-joining method with evolutionary distancesdetermined using the Jukes-Cantor method (D42, D43). Trees were rootedto the outgroup Escherichia coli (Accession X80725). The stability ofthe groupings was estimated by bootstrap analysis (1000 replications)using MEGA4. The percentages of 16S rRNA gene sequence similarity weredetermined for Sutterella C4-V8 products and Sutterella isolates usingthe EzTaxon server 2.1 (D44).

Western Immunoblots: Soluble proteins of cultured S. wadsworthensis(ATCC, #51579) were extracted and used as antigen in immunoblot assays.S. wadsworthensis antigens were separated by SDS-PAGE and transferred tonitrocellulose membranes. Membranes were blocked, incubated with eachpatent's plasma (diluted 1:100 in blocking solution), probed withsecondary antibodies [either peroxidase-conjugated goat anti-human IgG(Fc_(γ) fragment-specific; Jackson ImmunoResearch, West Grove, Pa.) orperoxidase-conjugated goat anti-human IgM (Fc_(5μ), fragment-specific;Jackson ImmunoResearch)], and developed with ECL Plus Western blotdetection system (Amersham Biosciences, Arlington Heights, Ill.).

Supplemental Materials and Methods

Pyrosequencing: 16S rRNA genes were amplified using V2-region specific,barcoded primers (E1) and products were sequenced at 454 Life Scienceson a GS FLX sequencer as previously described (E2). A total of 525,51916S rRNA gene (V2 region) sequencing reads remained after filteringbased on read length, removing low-quality sequences and sequences withambiguous characters, and combining duplicate pyrosequencing runs(271,043 reads for ilea; 254,476 reads for ceca). Binning of sequencesby barcode revealed similar numbers of 16S rRNA gene sequence reads perpatient (average # sequences per patient +/− standard deviation [SD],ilea: 12,320+/−1220; ceca: 11,567+/−1589) (see Table 19). Taxonomicclassifications of bacterial 16S rRNA gene sequences were obtained usingthe Ribosomal Database Project (RDP), Release 10, classifier tool with aminimum 80% bootstrap confidence estimate. To normalize data fordifferences in the total number of sequences obtained per patient, theabundance of sequences corresponding to members of the genus Sutterellaand all other genera were expressed as a percentage of total bacterialsequence reads. The abundance of Sutterella was also expressed as apercentage of total class Betaproteobacteria sequence reads per patient(see Table 19).

Operational taxonomic unit (OTU) analysis: For OTU analysis ofSutterella sequences, genus level classification from RDP was used tosubselect all Sutterella-sequences. Sutterella-sequences generated from454 pyrosequencing were aligned to the greengenes reference alignmentusing the Needleman-Wunsch algorithm with the “align.seqs” function(ksize=9). Pairwise genetic distances among the aligned sequences werecalculated using the “dist.seqs” function (calc=onegap, countends=T).Sequences were assigned to OTUs (defined at 97% sequence similarity)using average neighbor clustering. Representative sequences (thesequence which is the minimum distance to all other sequences in an OTU)from OTU 1 and OTU 2 were obtained using the get.oturep command inMOTHUR. OTU abundance by patient was expressed as percent relativeabundance, determined by dividing the number of reads for an OTU in agiven patient by the total number of bacterial reads obtained bypyrosequencing for that patient. Heatmaps were constructed using MeV(Version 4.5.0) using OTU abundance data from pyrosequencing reads.Heatmaps were drawn using Pearson's correlation as the similarity metricand complete linkage clustering. The upper limit approximately reflectsthe highest abundance recorded for any taxa in the heatmap (6%; red),and the lower limit reflects sequences above 0% abundance (0%; green);the midpoint limit (1%; white) is adjusted to highlight salientdifferences between the AUT-GI and Control-GI groups. Gray cells in theheatmaps represent instances wherein no sequences were detected for agiven taxa in a given patient.

Sutterella-specific PCR primers and probe bioinformatics: Evaluation ofgood quality sequences greater than or equal to 1200 nucleotides inlength revealed a total of 724 Sutterella-sequences in the RDP databaseat the time of most recent analysis (RDP Release 10, Update 27: Aug. 9,2011). SuttFor and SuttRev primers showed high exclusivity for the genusSutterella. Approximately 90% (692/768 bacterial 16S sequence matches)of all SuttFor matches and 98% (674/688 bacterial 16S sequence matches)of all SuttRev matches were specific to the genus Sutterella. TheSuttFor primer sequence matched exactly with approximately 96% (692/724Sutterella 16S sequences) of all Sutterella-sequences, while the SuttRevprimer matched exactly with approximately 93% (674/724 Sutterella 16Ssequences) of all Sutterella sequences in the RDP database. TheSuttProbe (nucleotide position 1011-1033 of S. wadsworthens is:Accession L37785) (Table 20) used for real-time PCR had low exclusivitybut high coverage of Sutterella-sequences (99%). The SuttProbe waslabeled with the reporter FAM (6-carboxyfluorescein) and thenonfluorescent quencher BBQ (Blackberry) (TIB MolBiol, Berlin, Germany).

Sutterella V6-V8 PCR sensitivity, linearity, and end-point detection: Todetermine V6-V8 assay sensitivity, Sutterella plasmid standards (seequantitative real-time PCR methods) were tested by conventional PCRusing the same conditions as for the biopsy DNA. Ten-fold dilutions ofthe Sutterella clone ranging from 5×10⁵ to 5×10⁰ were spiked into ilealDNA (25 ng) from a Sutterella-negative patient. We previouslydemonstrated that the ileal DNA from this Sutterella-negative patientcontains 16S rRNA genes from a broad range of bacterial phylotypesdominated by Bacteroidetes, Firmicutes and Proteobacteria, but does notcontain any Sutterella 16S rRNA sequences (2). The conventional V6-V8PCR was linear in the range of 5×10⁵ to 5×10² copies and had anend-point detection limit of 5×10¹ copies in the presence of backgroundileal DNA (FIG. 9).

Quantitative Real-time PCR Assay Details: For Sutterella-specificreal-time PCR on biopsy material, each 25 μl reaction contained 25 ngbiopsy DNA, 12.5 μl Taqman universal master mix (ABI), 300 nm each ofSuttFor and SuttRev primers, and 200 nm SuttProbe. The cycling protocolfor Sutterella amplification consisted of denaturation at 95° C. (10min) followed by 45 cycles of 95° C. (15 sec) and 60° C. (1 min). Fortotal bacteria real-time PCR, each 25 μl of amplification reactionmixture contained 25 ng DNA, 12.5 μl SYBR Green Master Mix (AppliedBiosystems), and 300 nM each of the pan-bacterial primers (515For and805Rev: Table 20). The cycling protocol for total bacteria consisted ofdenaturation at 95° C. (10 min) followed by 45 cycles of 95° C. (15sec), 56° C. (15 sec), and 60° C. (1 min). DNA from each of 128 ileal (4biopsies per patient) and 128 cecal biopsies (4 biopsies per patient)was assayed in duplicate. The final results were expressed as the meannumber of Sutterella 16S rRNA gene copies normalized to the average 16SrRNA gene copies obtained using total bacterial primers. Eightwater/reagent controls were included for all amplifications and theaverage copy number for water/reagent controls (background) wassubtracted from each ileal and cecal amplification prior tonormalization.

Western Immunoblots (Detailed Protocol): Anaerobic cultures of S.wadsworthensis (ATCC, #51579) were pelleted by centrifugation at 5000×gfor 10 minutes and stored at −80° C. Protein lysates were prepared fromS. wadsworthensis bacterial pellets using B-PER Solution (ThermoScientific, Rockford, Ill.) supplemented with DNase I (2 μl/ml B-PER),lysozyme (2 μm! B-PER), and protease inhibitor cocktail and incubatedfor 10 minutes at room temperature. The lysate was centrifuged at15,000×g for 5 minutes to remove insoluble proteins. The proteinconcentration of the soluble fraction was determined using the BCAprotein assay kit (Pierce Biotechnology; Rockford, Ill.). Proteinlysates (200 μg) in sample buffer (10 mM Tris-Hcl, pH 7.5; 10 mM EDTA,20% v/v glycerol; 1% w/v SDS; 0.005% w/v bromophenol blue; 100 mMdithiothreitol; 1% v/v beta-mercaptoethanol) were boiled for 5 min andsize-fractionated by 10% SDS-PAGE using a single large well on each gelto achieve uniform separation of proteins. Proteins were transferred tonitrocellulose membranes using the iBlot Gel Transfer System(Invitrogen). Membranes were blocked in 5% nonfat milk powder in TTBS(20 mM Tris-Hcl, pH 7.6; 137 mM NaCl; 0.3% Tween 20) for 1 hour at roomtemperature. Blocked membranes were transferred to a Mini-Protean IIMultiScreen apparatus (BioRad, Hercules, Calif.). Plasma from eachindividual patient was diluted 1:100 in blocking solution (650 μl) andloaded onto the membrane in the individual chambers of the Mini-ProteanII MultiScreen apparatus and incubated overnight at 4° C. Membranes werethen removed from the apparatus and washed three times with TTBS for 10minutes each wash. Secondary antibodies, either peroxidase-conjugatedgoat anti-human IgG (Fc_(γ) fragment-specific; Jackson ImmunoResearch,West Grove, Pa.) or peroxidase-conjugated goat anti-human IgM (Fc_(5μ)fragment-specific; Jackson ImmunoResearch) were diluted 1:50,000 inblocking solution and incubated with the membranes for one hour at roomtemperature, followed by three washes with TTBS for 10 minutes eachwash. Membranes were developed using ECL Plus Western blot detectionsystem (Amersham Biosciences, Arlington Heights, Ill.) and scanned forchemiluminescence using a Typhoon Trio imager (GE Healthcare LifeSciences, Piscataway, N.J.). Western blots were performed three times toconfirm reproducibility of results. Secondary antibody alone controlswere included for all immunoblots to control for nonspecific binding.Background adjustments using ImageQuant (Molecular Dynamics) wereapplied equally to all immunoblots.

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What is claimed is:
 1. A method of detecting a Sutterella sp. in asample, the method comprising: (a) selecting a Sutterella sp.-specificprimer pair, wherein the primer pair comprises a primer comprisingCGCGAAAAACCTTACSYAGCC (residues 31 to 51 of SEQ ID NO: 17), or a primercomprising GACGTGTGARGCCCYAGCC (residues 23 to 41 of SEQ ID NO: 18), ora combination thereof, wherein S is a G nucleotide or a C nucleotide,wherein Y is a C nucleotide or T nucleotide, and wherein R is an Anucleotide or G nucleotide; (b) contacting a nucleic acid from thesample with the Sutterella sp.-specific primer pair in a reactionmixture under conditions that promote amplification of at least onepolynucleotide amplicon, wherein the primer pair will prime a polymerasereaction only when the nucleic acid of a Sutterella sp. is present; (c)amplifying the at least one polynucleotide amplicon by a polymerasechain reaction; and (d) detecting the at least one polynucleotideamplicon, wherein the detection of an amplicon of a selected, knownlength is indicative of the sample containing the nucleic acid of aSutterella sp.
 2. The method of claim 1, wherein the sample comprisesintestinal tissue, feces, blood, skin, or a combination thereof.
 3. Themethod of claim 1, wherein the primer pair comprises SEQ ID NO:
 11. 4.The method of claim 1, wherein the primer pair comprises SEQ ID NO: 12.5. The method of claim 1, wherein the detecting further comprisescontacting the at least one polynucleotide amplicon with a probe havingSEQ ID NO: 19, wherein Y is a C nucleotide or T nucleotide, wherein R isan A nucleotide or G nucleotide, wherein W is an A nucleotide or Tnucleotide, and wherein H is an A nucleotide or T nucleotide or Cnucleotide.
 6. The method of claim 1, wherein the primer pair comprisesSEQ ID NO: 17, wherein S is a G nucleotide or a C nucleotide, wherein Yis a C nucleotide or T nucleotide, wherein R is an A nucleotide or Gnucleotide, wherein B is a T nucleotide, C nucleotide or G nucleotide,wherein V is an A nucleotide, G nucleotide or C nucleotide, wherein D isan A nucleotide, G nucleotide or T nucleotide, and wherein K is a Gnucleotide or a T nucleotide.
 7. The method of claim 1, wherein theprimer pair comprises SEQ ID NO: 18, wherein R is an A nucleotide or Gnucleotide, and wherein Y is a C nucleotide or T nucleotide.
 8. Themethod of claim 1, wherein the detecting further comprises contactingthe at least one polynucleotide amplicon with a probe having SEQ ID NO:13.
 9. The method of claim 1, wherein the detecting further comprisescontacting the at least one polynucleotide amplicon with a probe havingSEQ ID NO: 14.